The following is 0 true copy of my notes, Yolume III, labelled “Summaries” which were recorded during the interval 1951-1956 while | was in the Lederberg laboratory in the Department of Genetics at the University of Wisconsin in Madison. The notes were entered in intervals with some pagination and represent various types of summaries, speculatiuons, etc. | have numbered them sequentially in the upper right hand corner from page 1 to page 253 for convenience. There are a number of irradiation experiments (UY) plotted which have never been published. | remember communicating some of them to E. Kellenberger who may have giventhem to Werner Arber since | believe there are some similar experiments in Arber's doctoral dissertation. There are also some drafts of my own disseration. Items of possible interest are the handwritten notes of J. Lederberg (4/10/54) labelled “Remaining Questions” on pages 90-92; the typing bill for my disseration, page 155; 4 status report of the Lederberg lab for 1953, pages 161-195; some notes of JL on putting the stock book on keysort cards, page 197; a matrix by JL for transduction mapping, page 201; and an index to Volume I! of my notes, pages 202-206. This Volume is a hodge-podge and doesn't represent any temporal order - | believe page 253 is really ahead of page | which was a preliminary report leading to my dissertation. WA re M. L. Morse Webb-Waring Lung Institute University of Colorado Health Sciences Center Denver, CO, 80262 November 20,1986. . Summaries Research aM. L. Morse Gene tics Materials and Methods The principal cultures used are listed in table 1. In summary they represent three distinct matatd ons which lead to the loss of ability to ferment gakactose (Lederberg, F., 1950). The Gal,- and Gal,- stocks are the result of a single mutation to (-¥ in each case, while the Galo- stocks represent two independent mutations te (-) whose identity is based upon the observation that no(+) recombinants have been observed in more than 11,000 prototreophic recombinanta from crosses between them and upon the syiotyscus behavior of the stocks in transduction experiments. These three loci are closely linked to one another as indicated by the data in table 2, but the order of the loci is not specified. In addition, each of these loci is known (Lderbberg, E, and Laereberg, : J., 1953) to be closely linked to the Lp or latent phagé locus of E. cli K-12. Three alleles are known to exist at the Lp locus; (1) Lpt+t, overtly lysogenic and showing evidence of free phage in cross brushes with Lp® forms, and resistant to lysis by free lambda phege, (2) Lp™, not overtly lysogenic and showing the presence of free phage in cross brushes with Lp® forms, but resistant to lysis by free lambda phage, (3) Lp®, not lysogenic, and being lysed or lysegenized by free phage. More extensive delineation of the interrelatiouship of these loci has not been feasible until recently and it is hoped that with the aid of a new method of di‘ti neni shing the minus recombinants that mapping of this region may be accomplished. . Another lecua which has a direct bearing upon the problem is the locus contolling resistance to lambda~2, the lytic mutant cf lambda. This locus, Lpo, © has an epistatic effect upon evente controlled by Lp (Lederberg, E. and Lederberg, J., 1953). Thus a change from Lp,® (lambda-2 sensitive) to Lp", (lambda-2 resistant) results in a loss by the cell of the ability to adsorb lambda as well es lambda-2. Sensitivity to lambda by a cell therefore can be nasked by the presence of the r allele of Lp. The Lp, Locus is not clesely is—net—etosety linked to either Lp or to any of the galactose loci in question. Methods of cultivation and media used were as detailed in Lederberg, J. (1950). Liquid cultuivetions were in penassay broth medium, with or without added aeration, solid media used were of EMB base, either with or without added suger, or for crosses, @ synthetic form of EMB, EMS wae used. . High titkered lysates of cultures were prepared after tho method of Weigle and Delbrtick (195 ) by inducing lysia of penassay eon cells by means of irradiation with small doses of ultraviolet. The UV was adinistered to saline x suspehsions of the ceils and the cells subsequently diluted with 2X penassay broth and incubated wigh aeration until maximal clearing was obtained, Lytically grown lambda was obtained by infecting the sensitive cells by exposures to lambda prepared by the Lwoff technique, discarding the superia~ tants after the adsorptions ang resuspending the sedimented celle in mtrivnt saline broth. The NSB suspensions were then incubated wth aeration until maximal clearing was obtained, Table I List of pertinent cultures Culture Genotype w- - i ? W518 FM Lac, “Gal, Lp) *Lpo® =f, - - + 8 W750 FM Lac, Gal, Lp, Lpo well F'WLac, Gal, Lp,” lB, W902 FTL Th” Gal,” Lp; lp, W1210 FM Lac, Galo Lp) Lpo W1436 FT L-th"Lao, “Galy-Ip, *Lpo® w1924 FM Lac, Gali, Lp1"Lpo* +> on, >. W2175 F gal 2 Lp} Lp,° . . ie . w2281 ¥ M Lacy Galg Lp, “ipo - + W2342 F*Lac) “Gal"Lp, “Lp,® W2373 FH st"Leuc"Gal, “Lp; "Lpo® Table 2 Recombination between the Galactose Negatives Cross Minimum Number of Percent Prototrophic Recombinants (+) Recombinants FY Gelj- X FGal,- (1) 1500 0.13 o*@) (2) 65197 0.06 exe) (39853603 0.027 11620 . 0.06 F* Gal,- X ¥Gal;- 4588, os, 0.13 tye FA ¥* Gal,- X FT Galg- 2654 str 0.23 mounts of Results yard 0 af wild tena cultures are mixed with Galj-, When high titered lysate ae Galo- wat Gal; cells ani plated on HS galectese medius, results such as those in figura a are obtained. Since each of these mutations te gal- is capable of reverse mutation tne data shown in figure 1 have teen corrected for the number of reversions by subtragting tnis number as determined fram cantrol platings with no added lysate. Figure 1 chows that with increasing amounts ef added lysate thers is a Lixnsar increase in the number of galactose fermenting papillae per plata, In addition, figure 1 indicates tnat lambda sensitive cultures appear te be more capable of showing the effect of added lysate than lysogenic cultures of cultures carrying a nen-pleque-forming type of lambda. When lysates of gal- cultures are mixed with the various gal- cells and plated upon galactese medium results similar to these shown in table 3 are abtained. Each of the lysates of the gal- is capable of evo-iug galact-se fermenting papillae uppn plates spread with the other gai- cell types but not with plates spread with cells of its mvn type. The adility te wekneduce galt Clones in other gal=- bat not with cells of type corresponds te the differentiation ef thease gal- mutations dy PVecotibinabiaual analysis. Evidence ceuplemenating this ia ahown in table 4 which shews that the ability tr evoke papillae with cells of type is restored by reverse mutatinn. Presumably phenotypic reversicus can be at two types, reverse mutatic-n at the mutated lncus, and mateation ata gecend locus whose action waimicy tie actiru of the first gene. Reversians of this second claas should not be able to evote papillae frem cejls of type. Such reversions as the latter have unt as yet been investicated. - Pable 3 Interaction of Gal;-, Gal,- and %al,- e Gal,- Gal5- Gal,,- Wild Type miter (x10) - 2.42 4.9 12" 1,4 Cells Gal,;- Lp* (1) ge - 176" 43 a (2) 2 2 - - 405 Galo- Lp’ (1) ib 52 ll 43 - (2) 20 - 10 - 356 Gely- Lp* (1) 89 - 202 - 7 (2) 50 85 - - 417 (3) 47 - - 50 394 * Number of pepillae per plate, 0.1 ml lysate plated. Between 10° and 107 cella plated Table 4 , Restoration by Reverse Mutatian of the Ability to Transduce Previously Nontransducible Loci Locus 7 AdaSti on fall by) Reversion _None Reversion Lysate Gal,- Gal,* #1 0 6B | Gal.- Galg, #1 10 96 Gal, #2 6 552 Gal,,® Gal,” #5 39 204 Gal" #8 25 291 * Number of popillaeoper plate, 0.1 ml lysate plated Between 10 and 10 celle paased. @ Examination cf the other characteristics of the cells transduced to gal (+) by lyeh&’e exposure has uniformly shown no changes in any of them with the exception of the induction of lysogenicity in the lamada sensitive forms. Direct attempts to transduce other factors have been uniformly negative. A summary of the avilable data is given in table 5. In connection with the negative results in attempts to transduce xylose and lactose loci it shruld be noted that both xylose and lactosd containing media have some selective value for galactose ferganting clones. . Transduction in K-12 thus far haa been found to be limited to several galactose loci closely linked to the latent phage l-cue, Lp. These loci include Gal), Galo, Galz, Gal, Galg, and pessidly several more that have not as yet been Classified. The experiments reported here will concern obly Galj, Galo and Gal, although some observations on Gal; and Galg have been made. Hot ali loci contmolling galactose fermentation are transducible. Cne occurring in W2312 will be mentioned later, and another induced by copper treatment by Helen Byyers hag been found. The transductions described above have been effected by means of lysates perpared by the Lwoff technique of inducing lysis with a small dose of ultraviclet. Lysates prepared by lytic growth of the phage on a sensitive culture apparently have no transducing activity and have lost the transducing activity {ncluded in the inoculum. The inability of this type of lysate to transduce is demonsgrated by the results given in table 6. The necessity for lambda adsorption for transduction is illustrated by the results given in tavle 7. When the various gal- are found coupled with the F allele of Lpo, a conmblaskion which is incapable of adsorbing either lambda oF lembda-2, transductions are not observed. The presence of this allele of Lpy does @) not interfer in the capacity of a culture to give rise to &ransducing lysates transducrele aud the transducibdility of a gal- locus found coupled with Lp." ia demonstrable when a multable cross is made and a gal- Ip,” recombinant obtained. Recovery of the transducing activity of a lysate by the method of mixing lysate and cells on plates appears to be gool in the case of lysogenic cultures, the variation being less than two-fold over a thousand-fold change in the number of cells plated (figure 2). This is not the case when the added cells are lambda sensitive, the variation beiig in this a twp or three-fold greater over & similar range of cell concentrations. It should be noted again that t he lambda sensitive cultures give approximately ten-fold or more transductions at any cell densitiy, and that +H@%, the relationship of the activities on the two types of cells 1@ not known. the ratic of number of transductions to phage content of the lysates approximates 107? for lysegenic assay cells, about 1076 for sensitive cells, imtziztaxrizree Alternatively to mixing cells and lysate on plates the transducing activity of the lysates may be adsorbed upon celle and the cells then plated out on agar. Table & gives some indication of the adsorption of the transducing activity and also some indication of the adsorption of the phage under the same conditions. ranie gi | ee Effect of the Lp Locus on Transduci bility i Y dy tt eer Galactose Locus ig Addi $i on “Wilda Type Lypate Gali- Lp), Lp2® 1* 426" Gal,- Lp) Lp2 1 2 Gal,- Lp,* p,* 20 356 GalS~ Lpy* ipet 14 | 14 +. 8 Gal,- Lp, LPo 89 296 Galy- Lp,’ Lp5” 50 5? * Numbers of papillge per plate, 0.1 ml lysase plated Between 10° an 10” cells plated. rane #S | Other Loot tested but not found Transducible | - Locus _ Bamber of Experiments | Valtures Invokved __ Lac, 4 . W112 (ser or glyc) 1 W1678 Leuc 3 W1736, W1436_ Methionine 4 58-161, W811, W1821, W518 Xylose 3 W1821 s 1 W518. Prol ” W1692, W1920, W2062 Mal 1 W2071 : Hes, bom) | Table Action of Lytically Grown lambda in Transduction Seren Culture Mo Adgs ton lytic Lambda Lysate(2.4 x 1070 lambda/ m1) 228 _ W750 w 518 9 : 3 W2175 7 8 239 W750 2 0 - W518 13 8 W2175 6 2 254 W750 - 3** w518 - 6%" W2281 - g** W2373 - 6** W811 - 39%* erg of papillae per plate, 0.1 ml lysate paated. "0D 108-10 cells plated. re: *, es these peprilae picked aud streaked out all fouud - stable . WE pete” \ adsoeed om WitEE pol F wo foe Chpotnres. . Cabs Jeng laws with Gerahn . Carpe flee cotchD Fuh cotly Gren \ as fps dul yes ua gunned m NSB. Ger ted tr but etpoied Crthy . MASSACHUSETTS. NORWOOD, (NC,, > z < a = Oo v9 x oO Q a x wW Q 9 y BY FOUR CYCLES RATIO RULING, 4120 DIVISIONS) INCH PER 20 DIVISIONS NO. 32,226. PRINTED IN U.S.A. 2000 9 1000 ayetg dad ae[tideyz JO raquay Number of Cells Plated fable 8 Adsorption of the Transducing Activity from Lysates Adsorbing Phage Cell Percent Adsorbed Cells Titer Miter dst Ads. 2nd Ads.* 3rd Ads. X 10? X 107 Phage _ ‘Trans, _ Phage Trans, Phage _Trans. Galy- Lp*® (1) 2.5 0,71 60 79 50 41 16 46 (2) 3.9 0.55 52 33 - - - - Galy- Lp’ (1) 14 c. 10 - 79 (2) 18 16 - 72 - 56 - 0.0 (3) 14 ce. 10 - 97 - - es - Galo~ Lp” 18 6 - 35 - 33 - 0.0 Gal,- Ip* 18 6.5 = 45 - 100 - —00 * The supernatant from the first adsorption was decanted and an equivalent volume of fresh lysate added. Similarly for the third adsorption. Assays were made of the amount of material remaining in the supernatants. Titers given are for the phage-lysate adsorbing mixture. Tu the eLpevimeuts tuuelwiua, GCotg- Les GSIGy of the Sediment wast wéde Same wns tauces, Teh recavevy in freve Cotes Wor Mu tian loo - presumably be te. (ck Kaot wht achuh wos uudeveshwafed by me use of bre due oO (eu O55 OH cetls - Some of the papillae evoked by lysate exposure have a property which distinguishes tham at once from Spontaneous reversions. That is, they are unstable for galactose fermentation and segregate (~) cells ‘over many single colony transfers. The mature frequency of unstable transductions and the nature of the segregants will be taken up in-a later aection, {t is necessary to mention them now in order to consider the realtionship betwoen the transducing agent and the phage lambda. It is also necessary at this time fpr ad to mention some spectal coltures encountered during the analysis of the segregants mentioned above. These Spebial cultures are notable for the fact that they give rise to lysates by the Iwoff technique in which. the ratio of transduction activity te lambda plaque forming activity is much elaser to unity than is found in the usual cultures of K-12. These cultures will also be considered in a later section and it will suffice to say here that exposure ef a population cf gal- cells to one of these lysates can result in the transduction of several percent of the cells to galt. The data in table 9 indicates that when lamda sensitive cells are transduced the resultant cells and their gal~ sogregants heve for the most part become lysogenized. When Lp," forms are transduced thay also may become lysogenized, but much less frequently than sensitives. Hewever, these results may be misleading since the platings involve large quantities of i and it cannot be certain that lysogenization was not prior or subsequent to transduction. When the transductions are made with the special lysates mentioned above, Besults such as those shown in table 10 are obtained. Under conditions where one percent have been of the cells uum transduced to galt+ the transductions have become lysogenized, the same or Lp,’ , while the gal- cells in thts environment have remained lambda sensitive. , Table 9 Correlation of Lysogenization with Transduction © Locus transduced Lysate _ Transaducti ons . Segpangants _ Sad Lp Zonotype squrce Humber Percent Lp; Sumber Percont Lp, 7 @al,- tp,° wild 23 87 1 100 galo~ 24 95, ? 100 gals~ 12 58 0 - BAL 22 7 9 100 Galo- Lp," wild 13 85 13 85 | gal,- 20 95 20 95 galy- 23 100 23 ~—«- 100 wild 18 100 ~ - wild - - 28 59 gal,- - ~ Ady 86 galy- - - ko 83 Gal,- Ip) > wild - - 18 100 galo- - - 19 100 gal,~ - - 45 100 Galh- wy wild - - 29 3.2 galo- ~ - 18 5.5 Totals . 154 86 267 89 Table 10 Correlation of Transduction with Lysogenicity Using Lysates Giving a High Frequen Transducti Cells Post Number of Colonies Observed Exposed Exposure - to Cell Titer Gal- Gal+ Gal- partially lysed Total Broth 4.1x 10” 3280 0 0 3280 HF? lysate 3.5 x10? 2801 31(1.1%) 5 2886 ¥ titer = j.axtot 4 gieques pev ml. Table 10a inati on Colonies efter HFT Lysate gure Colony Number of Numbers, of Colonies of Fach Clase Col Examin Ip*® Lp Lp* Gal~ . 31 31 0 oO Galt 26 | 0 23 3 The occurrence of stable transductions among the various combinations of transductions possible is indicated by the data shown in table 11. With but six exceptions the difference between expected and observed fequency of stable gal (+) on the transduction plates is sufficiently great not to require statistical treatment. In setting out this data 1¢ has been asspmed that the only source of stable (+) on the plates is from spontaneous reversions and that the use of a no lysate addition plate as an indicator of the mumber of spontaneove’is adequate in this sense. It is notable that transductions _ duvolving gal, and gal, are nearly all stable and it will be remepered that lysates of these cultures have less papillae promoting activity upon: one * another than, ‘other cultures. These two loci are readily distinguishable oa crossing test and by use of the HFT lysates mentioned above. In the other combinations of transductions possible stable transductions occur, varying in frequency from less than one percent to more than 50 percent. The segregants from the unstable traneduetions adn be classified for locus by three separate methods: (1) by the 1 lysate by which they are not ‘transduced (transduction test), (2) by that Locus which cannot be transduced to (+) via a lysate (lysate test), (3) by allelism test in crossing (crossing test). In classifying the sggregants 4t will be convenient to refer to the origin of the locus by specific termf. Euterxhuuptypitexwtiixhexeitexs By homotypic will be designated the locus of the cell tranaigpduced to (+), by heterotypic will be designated the (-) locus (if any) of the transducing lysate, and by homo- heterotypic will be designated cultures with the loci of both trasifjduced cell and transducing lysate. | Since the order of segregation fram a transduced cell can not be specifie without micromanipulative means the analysis of segregants from a single transduction in its absence is without great significance. However, the data in table 12 indicate that a single transduttion can give rise to all three types of segregants, homotypic, heterotypic, and homo-heter otypic, fi ; Table # e Occurrence of Stable “ransducti ons ell Mumberss of Stable transductiangs Genotype ABZ : Seurce of Lysake - Wild “ype _Gal,~- Geio~ Gal yw 53-8 Gal -ip,® 1/93 1 = =O 0/56 201/30 289 #4 Gi) 130 oe § 6 49° Lp,” 1/46 2- @ - - - 1/92 0+ = . 26) 3 iS 234 weipyt ys4e = = YM 4 = = 12/27 27 a3(S4) os FF at’ 1:8 Galo-"Lpy® 0/46 15 0/214 27 - -«- - ~ 0/98 4% wee Se eT $ : ‘ 32.6 “Tp? 17/248 21- 14/83 61 - = - = 14/79 52 rH) 410 | Set > b tpt WB 6> 2/65 0- = = = = 5/56 0- +CS)M tH ‘ 4f.1 Gal,- Lp,* 19/835 383 29/72 72 11/472 20 4/128 22 - « FEES 07 set 9° Lp,* 41/573 133 51/96 96 - = « - - -« 224(976 67 3 50. “Ep, 31/320 127 = - 31/238 50 - - en qu) se Set u Exp't = number of stable expected no. papillae control no. papillae lysate plate Cbs. = number of stable observed = Ho. stable observed x no. papillae fransd. Reorder no. pap. in sample Note: A number of differeut lysates were employed. In the case of Gal.~ lysates, the first columa represents lysates of ¥902, the second column, W12I0. In the case of the Galj~ Lp, cells, the first is 4750, the second #2343, a prototroph derived from W750 @ Table 12 Segregants from a Single Transduction, tested by Transduction Test. The sequential ord the s 8 unkn Cell Lysate Classification of Segregants Genotype Source Homotypic Heter is H heter ic Gal) Gal,- ~ 2 1 & i but it can not be stated that the sceragpt one jin any sequence or if sequential. fhe analysis of single segregants from a large number of tranéductions was undertaken to clarify thie process. In the initial experiments the transduction tests were performed by mixing a portion of lysate from a culture of specific locus and the culture to be tested upon EMB galactose medium, but after the Av TRO discovery of the HiT lysates test for allele was by cross brush with lysates of this property upen the same mediun. , The results of _a-iarge-sunber of tests of segregants by transduction test ‘ts given in table 13 and a summary of the cultures in this table which were also tested by lysate test is given in table 14. The agreement between the two tests was complete, that is, a culture classified by vin first method as gal,- was also classified xy as this locus by the second testy A summary of the segregants which were tested by all three methods of determination 1s given in table 15. Agreement. between the crossing test and the other two tests was also complete. Some indication of the distribution of the segregant types, as judged by transduction test, can be obtained from the distribution data given in table 16. With regard to the crossing data given in table 15 4t will be noted that no crossing data for gal,~ scgregants ts reported or crosses of heter otypic segregants fron gal, transductions by gal,~ testers. Thistm because a suitable stock is not yet available. W2373, a hist” leuc™ gal,- made by transducking W1765 to gal,- has not been found “sufficiently fertile in crosses with mothe stocks te warrant its use. A new T"L~B,~ (also Het) gal,- aleo made by tranaduction to (-) may prove suitable. It should be noted that the number of protetraphic recombinants given in table 15 is probably lew by as much as 25 percent since in many instances only the smaxxexut plates with the smallest number of prototrrephic recombinants were counted in maxx experiments involving many replicate plates. j= Table 3p Analysis of Sezreeants by Transduction Assay. Summary, Nature of Type of Segregant criginal Homotypic"™ Heber otypic** Homp-heteratyoict*® Total . Transduction a Wild type on Gal~ 169 0 0 169 Gal~ on Gal- __240(85.4) 37(13,2) K(1,4) 281 809 (92-0) 37252} 4£6,88)} 450 4o7 * having the @al- locus of the transtinduced cell ** having the Gal- locus of the transducing lysate *** having the Gal- loci of both transinduced cell and transducing lysate. Table |4 Analysis of Segregants by Lysate Test. Summary. cher coment bptween Lysate Tests and Transduction Tests was Complete Nature of the original Homotypic Heterotypic Homo—heter otypic Total Transduction Wild type on Fale 21 0 0 21 Gal- on Gal~ 39 Iq 0 58 o If 0 74 Table \< Summary of the Analysis of Segregants by Transduction test, Lysate test and Crossing test, original Number of Classification by Transduction Segregants Transduction Lysate Crossing. test test test _B Homotypi Heter ot : How (+) Tot. Prot. No.(+) Tot. Rrot. Galo- --x Galy-Lp® 5 (1) Galy- Gal,- 0 2786 3 3183 (2) " " 0 2675 2 3471 (3) " " 0. 3485 23 5342 (4) " " 0 5952 1 1665 (5) " 8 0 5000 1 891 2 (1) Galo- Gal,~ 7 - 3102 0 1988 (2) " " 10 364 0 1187 Galo~ --¥ Galy-Ipt 4 (1) Galy- Gal, 0 16104 3. 1389. (2) “ " 0 $730 1 164 (3) * " 0 3358 0 202 (4) n 5 0 12848 1 171 art 3 (2) Galo- Galo- 1 11200 0 827 (2) a “ 6 10608 0 718 (3) " . 3 5000 0 hog _ Wild —=x Gal,-Ep® 4 (1). Galo- Gal 0 7805 N * (2) 8 ee 9 4992 (3) : " 0 106 (4) " a o 4552 N wala =X Galo-Lp* 4( 1) Galo~ Galo= 0 4070 (2) " " 0 5384 (30 " " 0 2072 (4) " " 0 6988 ‘Wild —-x Galj-Ip$ 4 (1) Gal Galy- 0 896 (2) 4 « 0 918 (3) " " 0 1134 (4) " " 0 863 @) (6 Table £1 Distribution of the Segregant Types by Transduction Assay Transinduced Source of Lys&&e cell Wild type Gal, Gal,-(4992) Galo-(W1210) Galy= Gal,- Ly” (W2343) 1f Gal,- = 18 Gali, 5 Galo- ~- no seg. found . Galg- Lp "(W2175) 20 Galj- 14 Gal~ - - 8 Galo= Pi ~2 Gal mo... 7 Gal) {2 Gal) —Galo-~: Lp}*(W1210) 15 Galj- — 19 ,Gela- - - oGai-- Lp,” 16 Galo>- = 20 Galo - - 21 Gal,- - 1 Galy- 1 Galo~Gal,~ | a Galj- Lp,* 20 Gal,~ nsf 16 Galy~ - - Lp,® 13 Gal,- naf 18 Gal),~ 17 Gal,- - 3Gal.— 2 Gala- Lp,” 29 Galy- nsf 15 Gal, - = usf = uo segregahts found SS Cultures giving lysases with the HFT property have been prepared for each of the gal- loci which have been given consideration to date. These cultures have the common property that each is derived from the transduction of a gal- culture by a lysate of gal,-. It 1s not known whether the transductions themselves of this type are capable of giving rise muty to HP? lysates cr uot, but the HFT stocks thus far obtained have been segregants from such transductions. Whether the transductions of galg- by other cultures gives rise to HF? segregants is not known, but one instance “la which the transduction of gal,~by gal,- resulted in an unstable (+# which had RFT preperty WOT HERA has been encountered. Sone idea of the frequency with which the HFT occur can be obtained from the following. If the case of transductions of pire gal, - by galo-» out of 28 gal,- segregants examined 4 had this property and of the heterotypic gal,-,one out of five examined was HFT, In the case of transductions of gal,,- by galo-, of 31 gal,- segrexgauts tested one was HFT, while of the three hetero- typic galo- tested one was HFT, In the above tests segregants which had been purified thr ough several single colony isolations were used. Since the HFT cultures segregate NYT. lines it is possible thattthe above estimatious are low. Attempts to obtain (+) cultures with HF? preprty by reversion of (-) have been unsucesyful in the limited attempts made thus far. This ton may be in part due to the fact that the HFT cultures segregate NFT Lines “since it was not known at the time of examination that this was the case anf the NFP reversions obtained could well have been from NFT components of the culture. The conversi-ny of a HFT culture EFEsHK to NFP is fairly rapid and the HF? cultuees are easily lost. On one occasion it was noted eA a@ culture which had been on stock slant only a few weeks had changed such that of ten colonies tested 4 were found to be MFT. The NFT cultures which remk are derived from HFT lines have not been investigated except in ime instancey, In addktion to NFT property (or possibly no activity at all) the segregants were inne case asmabire sgere Nope ne-faamennich UEEp wees demigted ant im qmwe otter iumimsre fury ware of a gal- type which was not transducible except by ax a lysate of an unstable gal(t+). In one case ( and the negative results in the other cases can possibly be explained > by the contammation of the HFT cultures with MFT cella) KA}°SX gal (+) reversions of an HFT culture were found to be unstable for gal(+) and segregated (-) which were of the same (-) type as the reverted locus. The examinati-n of more HYT cultures to to determine the relati-nship between duplication of certain loci and HFT property aud id in progress. | The lyeates of the HFT stocks which have been prepared thus far have not had high phage titers alth-ugh they have been prepared in a manuer which gives high titered lysates in MFT stocks. Whether this indicates a lower yield per bacterium of plaque forming particles or different conditions for induction is not known at the present. Preliminary experiments to determine the yield of HFT particles per bacterium are regarded with reservation since the purity of the culture with regard to AFT cells was not known. The HFT lysates have been used principally for allelésm tests. Transductions can alse be made via these lysates and the resultants studied. This has not been carried very far. The data in table 1% indicate that transductions by HF? bysates are not appreciably different from those of MFT lysates as regards occurrence of stable tramsducticns and distribution of segregants. The HF? lysates can be used for transduction from gal(t+) to gal(-) and have proved of value in creating new stocks. Table 18 lists some of the information available on the stocks transduced to (-). Since the completio: of the table gal)- and galo- Lp® o-LB,~ Ret Shave been prepared. The (-) stocks prepared thus far have been made starting with Lp® cultures. The resultant cultures may be Lp&, Lpt or Lp. In geueral the procedure has been to miy HFT lysate and cells on FMB(O) and incubate for 12-18 hours and then to streak out the growth and search for gal (-). on other occasions examination of single colonies from cell populetions exposed to HFT lysate has been used. Table 1p Transduction by HY? Lysates. “isbribution of the Segregants by Transduction Agsay Transinduced HYT Lysate Cell Genotype Gal,- : Galo- Gali- Gal,- py - lofali- 9 Gal,- 2 Gal, 1 Gal,= Gal,~ Gal- ip,” s Galy- : 8 Gel 27 an ae 1 Galo- Gal, - “15 Galy~ - Galy= by” not done * oot of & total of 18 transductions ( cr transductions and spontaneous papillae) analyze. The difference between the number of segregants reported and 18 represeats the number of stable papillae observed. © Table ( 7 @ Traneductions to Inability te Ferment “alactose Culture x Ip, ' Galactose “esul tant Transduced Genotype Locus Transduced Lp; Genotype Comment W1L85 Lp8 Galore Lp* or r 8 distinct (-) obtained from single colonies ? 2 distinct (-# obtained Galj- + ani r 2 distinct (-) obtained ¥1673 Lp® Galo + or - W1765 Lp® Gal,- 8 - W2252 Lp*® Gal, - r? 2 distinct (-) obtained Gal,- r 2 distinct (-) obtained ew) Separate mention of the cultures that were classified as double (-) by transduction test mst be made partially because the results are more incomplete and partially because they may offer some additional information upon the transduction phenomenon. Four such (—) have been obtained, three of the gal)-gal2- type and one of the galo-galy- typee- The evidence that such cultures are (--) is that they are transinced either by homotypic xor hetero- typic lysates but are transduced by wild type or some other gal (-). Lysates of these (--) cultures have been found to have little transducing activity regardless of the gal (-) tester used with but one excep ti oh. Whether this implies a failure of the phage particles to pick up a fragment of cell chromosome or whether the resultant thansduction is not phenotypically (+) through some interaction among the genes Concerned is not known. The exceptional case resulted in the recovery of each of the (-) making up the (—-) wauxrecsysrsd individually and not conjunctively. The homotypic locks transduced with this lysate was not recovered among the segreganta. As might be expected the (—) are more stable on galactose medium and have seldom been seen to revert. 2 Some experiments of interest have been performed with one of the (—) obtained. (It wa ortunately’a prototroph and the results obtained with must é it mtkx also be repeated and extended with auxotrphic strains. Although this (—-) was not transduced by uttkex , lysates of wither (-) singly it wae transduced to a lesser extent ( where a solid layer of papillae by a ratture of tue 190 with a (=) weuld have been obtained, less than 100 papillae were f ound),. In this ae : wos 4 case it, taken that the celle transduced to (+) had received two phage particles se meats with the addition of two (+) alleles in separate pieces. The cell that was transduced ty (+) may be represented as follows: 2 le and the resultant transduction as follows: a a ~27—-1t. -2t.~] ~~ In this case the extra (~) added in the segments are inferred from the results with transductions of single (-) in which the heterotypic lecus is recovered among the segregants. aus Segregation from this transduction in the absfnce of crossing over or exchange between chromosome and segments can result in three types of (-) segregante, | (1) meat ae (2) eee (3) 2-1 &2---1*. -2t-~1-- which would be classified as (—), (27) and (1~) presumably. With exchange between segments and the chromeome segregants with the (+) allebes weuld be found in the chromsome and subsequent segregation would yield( in addition to the types 2 and 3 above with the (+) transposed) the following types: (tb) e271 an (§) —~-2t-—-1"———s An additional type can be obtained if there be exchanges betteen segments. The order of frequency of exchange and segregation of the ebove types is unknown but on anaslogy with the simple tranifupions the first thewe mentioned would be expected most frequently, that is, loss of » segment is more frequent than exchange and loss of a segment.(This in turn is dependent upon the independence of exchange and loss) Examination of 2 separate segregants from one such transduction gave the following distribution of segregants by transduction test: 13 (—), 6 (17) and 5 §27). Since over 50 percent of the segregants were (—) it appears that when loss of a segment occurrs it is more likely to involve loss of both segments. The (17) and (27) found could be of two types, 2,4 and 3,5 above respectively. These types can be distinguished by means C of analysis of (+) reversions. In cases 2 and 3 the reversions will be unstable and segregate, and in cases 4+ and 5 they will be stable for galactose. Reversions were examined for their stability from each of the (-) obtained. All the (17) WEEE gave stable reversions and therefore were presumably of the ---27--1"—« type. Cf the (27) examined all but one gave stable reversions and therfore the two typee——-2°——-1?—» and 2 * were indicated with the most frequent being the former, Examinatioh of the ts (2) culture giving the unstable reversions showed that it xpaht did segregate (—-) cells but as yet it has not been established that it segregates (27) of the following type -——-2-—l*—* , The reversions of tty the type 2 (27) can be of two types and they should (perhaps) be distinguishable in turn by the segregsats that ty yield. Reversion of the form ~--2 yoy ---* should be expected to segregate (—) predominately ‘and veverstee ct ae the form —--2 ho shnvuld be expected to segregate (1) predominately. ea Reversions of the type 2 (27) appear to be of two types. From one type 33 segregants were obtained, of which 32 were (--), the remaining one 6 (27). The other type gave almost equivalent amounts of (2) and (—) and no (1°) thus far. The failure to recover (17) types from the wrx reverted cultures is disturbing but this may be related to elimination of the gal, locus in crossés. Presumably crosses between naan aa and —--2t—]"-—-« should yield a larger number of (+) than crosses between (17) and (27) of normal constitution when there is sucessful transfer of the segment through the zygote. these (+) in addition would be unstable for galactose. The culture used unfortunately is a prototroph and unless sucessful crosses between it and a Hfr strain can be accomplished the problem can not be attack from this aspect. ( Sucessful transmission of the eegnent thr ough the zygote was observed in some early experiments not related to the adore, 6G Examination of another (—~) has begun. In this case Gal, and aly, are involved and a croseable stock has been selected. There has been another complication in this case. That 4s when the culture was first isolated,and also in the case of a repaat test, it was not found to be transduced by either (27) or (4°) lyssates. Infeveral additional tests it has also reactive in this manner. In the instances where it was attempted to obtain transductions by mixtures of the two lysates it was found that the culture was transduced, to a lesser extant, by lysates of (27). Temctupxtustuxesrexcactrmmeserartiz It was thought to explain this incongruent result by postulating that reversions had occurred turing the growth of the culture and that in effect the cultge consisted of (—) abe} (47) contaminants. On this assumption the gransduetions of the culture would in effect be of the form (2-) ——-x (47) and the resultant transductions would be expected to segregate (4”) predominately. This was not the case, of the six segregants examined(from six separate transductions) 3 were (27), 2 were (0 and only one was (4"). This does not rule the explanation om but requires a frequency great eat of exchange between segzuent and chromosome for compatibility. \ Yxamination of this culture had progressed to the stage of isolating. a (4°) segregaat that gave unstable reversions as well as a yuxtet type which did not, at the time of writing. Not all of the Gal- cultures studied have been found transducible alz though the most frequently occuring (-) after ultraviolet radiation appear to be of this type. Three difinctly differ sat occurrence, of non-transduci ble gal- have been found. Two of these were induced by ultraviolet, and the third by copper exposure ( H. Buyers). ‘ne of the ultraviolet mutabis has been examined to some extent. The results are given in table 18. It appeare that this (-) is not transduced by any of the lysates and futher that lysates of it in turn traifyfuce all known transducible loci, but Galo with lowered frequency. QS) Table 18 Analysis of a Not-traneducible Galactose Locus in W2312 hy Tranaductiqn Asaay Experiment Plate Additions None : HFT Lysates AFT Gal. - Gal,~ Gal)- Wild Type 206 (1) o* o* o* 0 - (2) 0 8 - 7 9 220 (1) 0 0 0 0 - (2) 0 . o** o** ore 0 * mmber of papillae per plate ** NET (normal frequency of tranaduation) lysates used in these cases y fable 14 Activity of lysates of W2312 ou Selected Galactose Loct Galactose Plate Addition Locus | None W2312 Lysate Gal,- Lp* ips . 378 a7 ip” (220) 8 ? (221) 19 28** Galy- Lp* 17 74 Galg- Lp® 3 - 121 " mumbers of papillae per plate #9 12/12 examined were found to be stable Galt fable 15 Results of Crosses of W2312 with Selected Galactose Loci Selected Galactose Numbers Locus Gal+ Total Prot otrophic Rechmbinants Percent Gal+ Galo~ x 1 2112 0.05 Galy- F* 1 196 0.5 ® For the purpose of collecting new gal- and for observing the occurrence of tumnsducible loci two separate experiments were performed, Gal- mutations were induced in ¥1673 (glyc or ser)~ prol” and W1765 hist™ leuc” by means of ultravinlet. Table 19 gives a summary of these experiments. Reccurrences of both Gal,- and Galo- were found as well as a number of new loci and pessibdly several (-—-). Ne recurrences of Gal,- were observed. The effect of ultraviolet radiation on the transducing activity of lysates has been investigated in three experiments. The firet two experiments were concerned with MFT lysates, the last with an HFT lysete. The effect of ultraviolet upon NF lysates is shown in figure 2. With increasBing dose of ultraviolet there is a linear increase in the activity of the lysates on Lp* or Lp* assay cells until a survivial of the plaque-forming titker has become reduced about 1073. Thereafter there is a gradual decease in transduction activity with increastog dose. On Lpe there is a slight increase in transducing activity ant then a gradual decrease. The maximum reached by the lysate™ on Lp* or Lp celle is about four times the maximum reached on lp® celle. In performing this experiment about 16° Ip® assay celle were used, since figure 1 indicates that this number of cells may {nitcate only about x one-thizd to one-fourth the number of trensductions actually present the Lp® assay is probably thet much low. fhis then would suggest that the absolute number of transductions 1s approximated uponp® cells when a sufficient mumber of cells are used and that the action of ultraviolet is to increese the assay pn Lp* or Lp* cells to the level of the absolute number present. In connection with this it should be noted that survival of the transducti onexsuxy Lp® is still about 0.5 even at the extreme doses used. From the above it 4s suggested thet the action of ixsxtes of ultraviolet is weveral fold. Fires and most rapid fs the destruction of plaque forming activity a Lp* cells. Secoxmly, to destroy that property of the ag receds Bede che phage which causes them to pe"excluded" by lysogenic celle, and thirdly to destroy & oy, i - Aah Oe, Table Transduction Assay of Some Galactose Negative Mutana&s Induced by. Means Ultraviolet . Possible Culture Mutant Transduced by HFT PESHEMIE Treated Designation Gail,- GRTo— Gal)- Genotype W1673 Lp® W231o 0 + 0 Gal, -Galy- W2311 0 + 0 " " W2322 0 oO 0 nontransducible W2313 + 0 + Gal>~ Ww2314 + + + Gal - W2315 + + + Gal_- W2316 0 + + Gal, - W2317 0 + 0 Gal, -Gal,~ W2318 0 0 0 nontransducible W1765 Lp® 238-2 0 0 0 nontransductble pang + + + Gal _- 238-6 0 + + Gal,- 238-8 + + + Gal_- 238-10 + + + Gal,- 238-11 0 + 0 Gal, -Gal,,- 238-12 + 0 + Gal, 238-13 + 0 + Gal 27 G the transducing activity itself, perhaps by destroying the adsorption of the phage particles. The effect of ultraviolet on HFT lysates is similar to that of UV on KFT lysates. The increase in transducing activity with dose in this case ig not as great as with NPT lysates. A maximum is reached that is approximately equivalent to the plaque titer of the lysate which suggests that plaque and transducing particles may be the same but that appearance of a particle as a plaque exchudes its appearance as a transduction. Platings for plaque formation on EMB galactose have not indicated that one particle can function in both capacities but the appearance of a plaque might be obscured by papillae formation. The sum of the activities (maximal) of the lysate on the two assay loci is 2-3 times the plaque sktkxmx titer, which may be an indication that the activities are confined to a single particle. The occurrence of transductions with Lyp™ genotype has been noted with this lysate, and the equivalence of plaque and transduction titer might not be expected on the aswumption that in these cases the effect was accomplished by a defective phage particle which would not give as well as to rise to plaques 3F lysogenization. (This would reqbhire that Lp’ genotypes were the result of such defective particles rather than of a defective act of lysogen- 4zation.) SOON O t 9 “oogrh OM F ag ‘O09 N|3OZ131I0 3N3ONF ” q ayes lt ‘S¢n NI JOWW “OOoOrR OM YY SCHRTELT a7 9OR O H v “ N Oo Suu p4gonps tea e4 HON! 43d SNOISIAIO OF K S3154A0 GS DIWNHLIaVvVOOT-IWSS UBdvd HAVYD NSOZL3IA OCISI-OPE "ON (< v “gor on 2 ca ” 10 Ultaviolet Dose in Hiuates s Ss — nt TOONnOM ¢ g 7qns masp mip earl “fhf AAs APQUIAN } ‘O90 NAOZLIIG 3N39N3 , HON! Yad SNOISIAIO Zi KX S3IDAD G JINHLIaV904-INSS N3dVvd HdV4eH NADZLIIA SIS1-Orvre ‘ON gre pla ' S) Gace ad gerves ted as 2 uv. 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Me oak pa Cte vs poarege wih ue Cut “| Gum ner paint = da S GENETEC TRANSDUCTION IN ESCHERICHIA COLI By’ MELVIN LAURANCE MORSE A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of DO6TOR OF PHILOSOPHY UNIVERSITY OF WISCONSIN 1955 f TABLE OF CONTENTS log Introduction - \ Experimental results -\, \,7 General observations on tasnsduction” Observation on galactose negative cultures- | 4 BAe de of lysates of wild type awhile Cultue: ior of lysates of galactose negative Halle cuties - 1 % br of lysates of reverted gAhactose negative delle cul hues Considerations of the method of assay of transducing activity ~ e The necessity of lambda adsorption for transduction - ] YHELABHERKEIX AF AL SHA ELE XT EXSE AR GABELID EXEIO BEEN SEAR The activity of lytic lambda—- 4 fhe trensfemmed—-oebts franvducthon clenes—4 i dncidence | of lysogenicity in the transduction clones derived from Lp® recipient cells-\o Existence of transductions stable for gatactose fermentation-|?~ The segregants from the unstable transductions -14 Galactose negative cultures giving lysates with HFT property-/ 5S Experiments with lysates giving a high frguency of transduction- 7 The relationship of lysogenization to transduction -! % . The interaction of Gal, and Gal, (Position effect) -\4 The action of HFT lysates on Lanbda~2 resistant cultures -2© Crossing behavior of the transduction clones - J} z Galactose negative cultures that are not transformed by lysates -2 a ~ Disenssion ~ ol. }- Sa Materials and methods ” 4 e- yWwrvevyan dam cya. Summary - 32 ~ Bibliography -3 | Figures ~ Tables OO @ INTRODUCTION Exchanges of genetic matérial between bacterial cells can be classified into two main categories ( Lederberg, J., 1954). The first category is exemplified by the recombinational process found in Escherichia coli K-12 by Tatum and Lederberg (1947). This form of gentic change includes a syngamic process, that is, the conjunction of large blocts of genetic material, and there is evidence of linkage groups, linearity of genes, and requirement for intact cells ( Lederberg, J., et al, 1951, | Lederberg, Je, 1954). | Under the second max main category are found the exchanges where one of the participating cells is not found in intact form, but whose genetic material is presented as a solution or suspension of J particles much smaller tha the cell, —— This category has been given the general ‘title of transduction (Zinder and Lederberg, 1952, 4. Lederberg, 1954) J and is readily subdivided into two classes on the basis of sub of thansduchor- the vector of recombination. The first clase is exemplified by the pneumococcs transformation sy stem, (Austrian, 1952), where. the genetic changes are , prought ; {D - about by means of purified preparations of desoxyribonucleic nein In the second subclass the genetic changes are mediated by Bacterse® viruges or bacteriophages ¢ Zinder and Lederberg, 1952, Waianae TORRES iq Pome, syn aw, “WORD. In contrast watt Ll lee ater cng : genetic transduction usually results in monofactorial genic changes, although dual changee have been noted ( Stocker, Zinder and hederbere, 1953. Hotchkise, 1954). The frequency of occurrence of these exchange processes among the uw | | ‘various genera of bacteria is not known, Genetic recombination of the E. coli * 2 + ‘K-12 type has been observed in about 50 additional strains of E. coli of over [Ons Sim low te 2000 Santas (kederberg and Tatum, 1953). fransductim rinse atta — © pneumococcal teecmetersetst=a— ime have been observed in flomophilus infl infl nensag A? ~@ ( Alexander and Leidy, 1951), Weisseria menigitidis ( Alexander and Redman, 1952), and Escherichia coli ( Boivin, 1947). While strains of BE. coli are reported to show syngamy and traneduction, ESUELIEK Boivin's culture has been lost and farther studies with it are impossible. Att8mpts to transfer genetic material via desoxyrivonmerst: acid preparations in E. coli K-12 have been unsucessful. ( patchly, 1951). In Salmonella, Zinder and Lederberg (1952) demonstk&ted phage mediated transductions but failed to show the occurrence of syngamic recombinatéon. Thus, of the three forms of recombination considered, no one culture has previously been observed to exhibit more than one of the exchange processes. It is the purpose of this thesis to describe a limited system of transduction in BE. coli mediated by the lysogenic phage of strain K-12, lambda. The occurrence within the same sgrain of syngamic recombination and of phage mediated transduction promises to improve our underatanding of both processes. (3) @ The principal cultures used are listed in table 1. In summary MATBRIALS A.D METHODS they represent mutations at three distinct loci which lead to the 1688 of ability to ferment galactose. Such matations have been obtained by irradiating galactose positive cultures on an indicator medium, EMB galactose agar. The different loci have been atstinentenothy intercroasing the various stocks and. finding galactose positive recombinants in certain crosses (Lederberg, E, 1950). *he Gal,- and Gal, stocks are the result of a single mutation’ to (- ) in each case, while Gal. , stocks ‘represent two independent motations to (-) whose identity is vased. upon the observation that no galactose positive recombinants have been observed in more than 11,000 prototrophic - recombinants from crosses between them and upon the synonymous behavior of the stocks in transduction experiments. These three loci are closely linked to one ‘another as indicated by the data in table 2, but the order of the loci is not specified. In addition, each of these loci is known ( Lederberg, 3 and Lederberg, a 1983 ) to be closely Linked to Aa Le, latent phage ) Locus of 3 col K-12. Tiree alleles are known to exist at the Lp locusg ¢ (2) Lp’, ¢ overtly lysogenic am (showing evidence of free phage in cross rashes with Lps forms} and resistant to lysis by free lambda phage, (2 2) Lp* ‘f hot overtly lysogenic Ss Ee seer eee, wot! (Peststant to lyste by free lambda i> rhage, (3) Lp 8, not lysogenic, and — lysed or lysogenized by free hage. \ At least two other loci affect the interaction of lambda with E. coli E-12. and are scored by resistance to lambda-2, the lytic mutant of lambda, One of these shows a coincidence change in maltose 4h fermentation. Both mtations result in a loss by the cell of ability either to adit lambas or lambda~2 regardless of the state at the Lp locus. Methods and media were as detailed in Lederberg, J.(1950). Liquid cultures were in penassay broth, with or without aeration; solid media were of EMB base, either with or without added sugar, or Dikco nutrient agar with 0.5 percent saCl. Vor crosses, a synthetic form of EMB, EMS)was used. | . High titeréd lambda phage lysates were prepared by two methods. The first ank most commanly used was that of Weigle and Delbriick(195/) in which induction by ultraviolet radiation (UV) is used. The UV was administered to penassay grown cells resuspended in saline at a density of about 10? per ml, After irradiation the cells were diluted with double strength penassay broth and incubated at 37C with aeration until maximal clearing was obtained. " Lytic " lambda was prepared by infacting lambda sensitive cells with UV-induced lambda; the infected cells were resuspended in nutrient saline broth. These suspensions were then incubated at 37C with aeration until maximal clearing was obtained. Lysates prepared by UV 10 per ml, whereas the lysates prepared induction had titers in excess of 10 by the other method had slightly lower titers. Unless otherwise specified, the lambda used in the following experiments was obtained by UV induction of lysogenic bacteria. Crosses were performed by mixing % salinre suspensions of penassay grown cells either before plating on the EMS synthetic medium (usually with added galactose) or directly upon the plates “@ in which the culture is atreaked screee either phage or phage sensitive Teats of sultyres for phage reaction vere by the cross brush method cells to ascertain whether or not 4t ‘earrying phage or sensitive te phege ( Lederberg, M% ani Lederberg, %% 1953). . lysates qving 4 " Pransduotion aseaya were made in the case of <2 normal, hus frequency of transduction Snes by adding 0.1 ml of lysate to the sppropriate cells on EMB galactose agar and incubating the’ mate for a 48 | hours, A separate plate with 0 lysate bddod. served as an cttinete in ether cases of the amount of spontaneous reversion occurring, ox, the lysate was spread only apen one-half of the plate, With tn lysates elving 5... Oa (Her) high frequency © of frenaGngstonsthe lysate was eross brushed cerns oe on the cells, ry pa lis He tosts phage sensitivity. x © EXPERIMENTAL RESULTS General @deervations on transduction of a number of loci selected at yvaudom t for ability to - Tests he eave negative be transduced #4 results (table 3). ‘The tests for thansduction of the auxotrophic markers were performed by adding lysate to cells on minimal medium, the tests on fermentation markers on EMB medium with the appropriate sugar, Agdis was performed “The teat for transduction of streptomycin resistance ey growling the “G- addition = © | : ne roe oe Glactose negative cultures unable to fermert an additional ‘weer carbohydrate such as lactose, xylose, and arabinose ( Ee Lederberg, unpublished) will give apparent transductions wheh plated with phage | on media containine these substances. Such apparent tnpfpauctions are , “hot ‘for the fermentation of the carbohydrate in the medium, but for wih £4 veh * palactoge: fermentation,’ ainse: after purification: “the” ‘traneductions | , ” Slonee’a are ound only galactose positive. Media containing these substances . . Veet ae a ke ney tee Oh Lotaeg have some selective: action’ on galactose fermenting clones. ; st dheghee. ek ote ce whew oe, bese d feces Bo SI Stk Ee ALS ee wee bh ADV anee in the mumber of galactose fermenting papillae are observed (table 4), The number of galactose fermenting clones is proportional to the amount of lysate added (figure 1). Since eack of these mmtations to inability to ferment galactose ia capable of reverse mtation the data mast be corrected, {n-each—case. This haa been done for the data in figure 1 by subtracting the mmber of spontaneous reversions as determined from control platings with no added lysate. In addition to indicating proportionality, the data in figure 1 indicate that the cells show the effect irrespective of the Lp genotype of the cell, and that O. rk dambda sonsitive om are more capable of showing the effect of added lysate than lysogehia chkltures, See AY: ot reJ ae of _galactose negative Sakis. | When lysates of galactose negative cultures are mixed vith the various galactose negative cells results similar to those shown in table 4 are obtained. With the possible exception of the interactions of Se and Galys seach of the lysates is capable of evoking galactose fernenting popilise upon plates apread with nemhonologous negative - celle. with the umal lysates Gals Cady, interactions are erratic, sone- tines giving stentficant differences detween control and Lysate added plates, sougtines not. This lateraction will be dealt with in more detail in a later section, it will be eufficient to state here that pos drvl such interaction does not produce elenes that are phenotypically SB. ot Mese loci The differentiation aby lysate interaction corresponds to the differentiation Apppest-b6ek by recombinational analysis. Actiuit - 3, Babotte of Lysates of reverted Balactose negative cultfires. : Reverse mation restores the ability of lysates of a galactose Md 2s hoe _-_ a FP oo daw Rte Seyi St wes boat 4 OR PRESS . PAR Ye on . ‘ : Ae . , ay wee 3 FP oseae Be eat eas aS as an Let ha rn ee ; 1 4 ve ‘ oR Ra eR ne ES geegtoos. Minte: reversals should be able to evoke: pepttiee | fron. cells of a a original matant type only in the improbable: event, that they | are located | _ in the restricted genetic. sognent, thet appears to be capable ¢ of genetic ee t4 ay oy Tee SR EE aT 2 ee at ee RE Te ee ee . e eet oo Mae wag ek ae Kg dpe atin nae hha Shee Aue wees a ne My ME GALA Sha ELEE eae 6! ef the transducing activity of a lysate by the method sakithpcles of mixing lysate and cells on the plates appears to be gam in the case of lysogenic cultures, the variation being lesa than two-fold over a thousand~fold change in the number of cells plated. Cell concentrations ©) oPTIMAL Detween 5 X 107 and 5 X 108 appear to give Ee cscctson of lysat: activity. When the assay cells are lambda sensitive the variation is two to three fold greater over the thousand-fold range of cell values from 106 to 107, with increasing assay values as the number of cekls increases. Since the ratiog of phage particles to transducing particles in a lysate is very large the interaction between lysate and sensitive cells is compl ex, axa xan with. the great probability that the inactive phage particles Rion influence the expression of the transducing particles. | a ee ee ee The ratio of transductions to phage content of the lysates varies, -6 approximating 107? for lysogenic assay cells, about 10 ~ for sensitive cells, that is, about a ten-fold difference in efficiency. The necessity of lambda adsorption for transduction The necessity for lambda adsorption for transduction is illustrated by the results given in table 9, When the various galactose negative cultures are lambda-2 resistant, a combination which is incapable of adsorbing either lambda or lambda-2, transductions are not obtained. The ability to transform a galactose negative locus found coupled with lambda~2 resistance is demonstrable when a suitable out cross is made and the galactose negative lambda-2 sensitive recombinant obtained. Lambda-2 resistance does not effect the ability of a lysogenic culture to give rise to phage and transducing particles after UV induction, g oot vty q) he palfterot lytic lambda. * ~ tHe transductions described thus far have been effected by eans of yeates Prepared, by the ultraviolet intuotion technique. Lyeates prepared by igtic grovth of the phage ona sensitive calture pparently havé no transducing activity end have lpst the transducing activity included in the starting Sam phage {nooulun (table ®): a JN The pasihe chm gfgnes With the exception of the Lp locus in the case of lambda sensitive cells, no changes have been observed in any of the other genetic characteristics of the transformed cells. Many of the galactose fermenting clones produced vy transduction are different from the spontaneous reversions in their instabilfty for galactose fermentation and in some cases for lambda reaction. That is, they continue to segregate galactose negative clones in the course of many serial {solations. In addition, in the case of the transductions with Lp” reaction there is seer dgétion for lambda sensitivity with segregation for galactose fermentation. Lysates from unstable transduction clones also differ from lysates of galactose reversions: in the former the ratio of transductions to plaques 4s much closer to unityy (table 8). Lysates of the cultures unstable for galacbose fermentation when prepared in the manner of the other cultures © @ have lower phage titers. The reason for this is not known but the production of phage in these lysates is being studied further, With the exceRtionyl of transductions formed with wild type lysates, the transduction titer of these lysates is dependent on the genotype of the assay culture. When portions of these lysates are cross brushed on galactose negative cultures the intersection of the streaks is converted principally - to galactose positive growth because of the high frequency of traneduction (HFT), The problem of the HFT lysates will be dealt with in more detail in a later section. Incidence Sfclysogentcity in the transduction clones derived from Lp® recipient calls, When MIT lysates are used in transductions to Lp® recipient cells, about 90 percent of the resultant transduction clones are lysogenic (Ip’) or Lp’. There is some slight evidence for lambda sensitive transductions, but these putative transductions have been found stable for galactose fermenattion and it has not been possible to distinguish them from spontaneous reVersions except by their frequency of occurrence, @ When Lp” cultures'are treated with lysates a small fraction (3-5 percent) of the segregants from the resultant transductions are lysogenic whereas it had not. been possible to lysogenize Lp’ cultures with previous methods (Lederberg and Ledenberg, 1953). The high incidence of lysogehicity in the transduction clones may be misleading owing to the excess of phage, and it cannot be ascertained whether lysogenization took place before, concomitant with, or after transduction, by the NFT phage. In the section on HFT lysates the rdefftonentp between transduction and lysogenization will be shown more clearly. The segregants from the transductions with Lp" reaction are Lp’, while the segregants from the Lp” transductions are Lp” and Lp’. In speaking of the Lp* reaction it shpnld be noted that the classification of Lp” is more subject to quantitative considerations than the other alleles of Lp. The two cultures (W1924,W1027) derived from sources other than transduction that showed no plaque forming phage in cross brushes with sensitive cultures gave plaque forking phage after induction with ultraviolet radiation, The amount of phage was greatly reduced over that obtained from Lp’ cultures under similar conditions. These two cultures were obtained after separate procedures, one from an ultraviolet irradiated Lp’ GW1tti@, the other from an Lp* culture treated with lambda (KE. Lederberg, unpublished), Both were stable as regards their lambda reactions, The Lp” clones observed after transductiion have not given plaque they | forming phage after U.V. exposure, but differ from those which have given phage, by instability at the Lp locus Whether the bransductions with Lyp* reaction are the results of heterogeneity among the phage particles, the cells, or as the results of a "defective" > act of lysogenization is not known, but presumably the problem could be investigated by statistical or | Existence of transductions stable for galactose fermentation. Te evidence for the occurrence of stable transductions is the increased number of stable galactose positive clones a on lysate platen gamer “than eqpucrtet Geom combust piakines{ tabséa 4). Although the increase could also be explained on the assumption of a change in oe fayorin Spmrancout reversions ©: tinding that molt oo Chen ave alts ; selective condi tions 6 fac eated, lysates ( 56C for 30 minutes y : “yr peme tn Phe Coan | hh or filtrates of galactose positive, lambda sensitive cultures gave 7 et no increase in number of papillac, suggests. that change in selective Gud conditions is ‘not the case. THE S&6¢téGanre From THe UNL TABLE TRANS OUCH NS ae — The non-fermenting segregants from the unstable treneincttiy et negative cultures against tyeates of the segregants,(3) by cresses wih known galactose negative types. In Classifying the segregants it will be convenient to fefer to the BSERSKARERXSEXERS parental source of the negative allele or alleles by generalized designations. By idiotype is meant the genotype of the recipient cell parent, by allotype the genotype of the donor source of the transducing lysate, Amphitypic will designate cultures whtch at some joci are. idiotypke and at others are allotypic. le Unstable. or. segregaine stocks,.as will appear, are heterogenotes and the underlying state is described as heterogenic to distinguish it from [| euploid heterosygoeis for satire gence, oo aa . wo | = For further analysis tt will ultimately be desirably * rsp ee rut single cell pedigrees. The following observations on wy } cues Age MADE Yaolations, with due regard to the complexities of colonkey fo three methods, and ‘some cates (table 10) by all mothods., Tables 1% and 19. set nt Behe esos oo Boga Bay coe: ‘ a Present wmitmarios of the analysis ag transduction zeeipients and ag #. felgee PRAM 8 apo nay cE Eee May eee Cae RET : for this purpose stock is ts aratighg fe ON SS a Shen etal ot tant See eeegts. brag is, a culture Slassified: by the. first method mas: = aly was alec classified as: this wits by. the other tire teste, : pe no mo | ree segregint . re classifi hi ty against Iyeaton of known cultures, 3 ¥ re a 3 - Gat,-, and one was Gal,- Gal,~. “The former Were Prototrophi and it was not possible to exanine their behavior in crosses, the al, Gad, culture is Croseable but has not veon tested Rereteceiidl a0 yet. ce Because ot the Gal;- ean, interaction it is not Feasible to test loc any of the anph typie segregants weing only. the threes 80 far considered, . Attempts were made te analyse the amphitypes. further oy the action of. their lysates on an m addi tional locus, Gale-. Lysates of the two Gal, ~Gal,- were plated with cells of a alge culture, Both lysates had little action in producing papillae . (This perhaps might have been expected since ated, ae 8 @al,- fauna on, Galg-). Several unstable galactose fernenting clones wétte ‘obtained from each Interaction, Aoverer, and a amunber of: segr egants were tested. or 16 segrogante trom the ‘transtuotions by the lysate of one aephitypic culture, 15 were dal, - ® and one was ‘classified as Gal)- Gal,-. From the action of the lysate ef the second amphitypic culture five Gal,- and two Gal,- segregants vere obtained. Although both lysades @) wag ale ofiels transmitted Gal,- and Galo-, confirming the existence of these Gm in- the: parental. enltures,: the failare to recover the idiotypis Galg-~ locas among the negregants is disturbing. Beyond the fact that Gal, is @ locus transduced by lysates nothing is known of. its behavior. MEE a teat) Al th th ic cultures ar % tranef ed to wild. ough o amphitypic, ef are no orm we bind , 4 type. by, the. action of a Miisiaahe: pure. ven . cE. the. _stattatios, of, the interaction of cols and, Lysate hate not OS ik det _ deen investigated, wat the greatly reduced pander: of transinetions produced gril eed ¢ y the mixed lysate is expected on the. assumption of indepenient interaction a ‘Dehween the cells and each of the. transducing activities, . The tranesductions produced by the action of. nixed lysates on . amphitypie segregants appear to be less atable than traneiuetions of caltpres ~~ oe | Saenecative at a single galactose locus. In addition they give rise to _ Sintermediate" segregants in whieh only one of the éwo transducing activities has Been los} from the WMA clone. grese "intermediate" segregants in turn give rise to Teregante from which both transducing activities have heen lost, t ‘ Under the section on transformed cella itk was noted that in lysates of the unstable galactose positive clones the ratio of transduction titer oe eck PE MARKABLY 0 Kew plague titer was gubye, high ny, Aenea herent apenarre— Ye te sn ceenenaTE clamped these cultures were not the first ‘to cive HY? lysates. In the course of eine ergata fron A lysates traneduotion, by means of lysates of then, several excepite waitress were of transduction. aq ee for the ee =a bn meee eet . ilar these exceptional culture no aifrerent: fron -. other secregants. vere: ‘unstable - guachy Regarding the Latter inatabtlity, HFT : cultures which were negative ‘at a. for this property ond uastanle, on, rare. oncastons ‘for ng single locus segregated MFT segregants. that were negative at this lecus | and pip Miah ‘wane negative at sh addi tional locus as well. In most... instances , hovever, the BFE segregants were of > ome nesative Sea Kr THe SAME os the parent galactose negative HF? culture. the galactose positive yeversions of the ‘ae cal tures that have been studied are still capable of giving HY? lysates, Tat are unstable for galactose fermentation, , The galactose negative sogregante fron the reverted ne ‘cultures are at, are elther negative at the same locus as. the original negative HY? segregant, or negative at this locus and negative which proved tole th thd ovig (nad - at an additional locus, one, was th tadotyst locus in the fornation Zz Ib © of the traneduction clone. The galactose positive reversions of these segregants are stable. A charactersitic HFT culture has been obtained for each galactose uagt negative as well as for wild type. These cultures were isolated initially by making lysates of random segregants from heterogenic transductions and assaying the lysates or the appropriate cells. This method is laborious and inefficient. To assist in the isolation a nore rapid method was devised. Random segregantZ colonies were picked to small volumes of water or broth and a samples of each suspension were then spotted on an EMB galactose plate spread with cells suitable for the detection of the HFT culture desired. The plate upmkked was given a small doee of UV (about 10-20 seconds at 50 cm froma Sterilamp) and incubated for 24 hours. At the end of this time HFT cultures were usually detected by the raised welt of galactose positive growth where lambda produced by the induction and lysis of the HIT culture had transformed bacteria of the background film of growth. _ The incidence of HFT galactose negative emkutezx cultures ig not high. Of 67 segregants tested, 7 wére found to be capable of HFT lysates, The true frequency might be higher than this, since gurified segreganfs were examined and there was opportunity to pick .FT segregants from originally HFT clones. Cultureskux giving HFT lyeates that are pure for a particular galactose negative allele are suitable for allelffin teste of unknown gelactose negative cultures by the cross brush method. @ v Experiments with lysates giving a high frequency of transduction Although the HFT lysates have not yet been obtained with phage titers comparable to. Mrs lysates the titers have been sufficient for transforming a large fraction of a cell population exposed to them. The largest fraction of transformation observed thus far has been 12.5. percent of exposed cells, but in most experimexts the fraction has been between l and 5 percent. _ The use of HFT lysates has permitted the study of several problems not attackable with NT lysates. One of these is the relationship of trans- duction to lysogenigation with the phage lambda. Another problem is that of the interaction of Gal, and Gal,. Both of these problems will be dealt with in the next sections. With pre lysates, transduction was experimentally feasible ag X only whan a galactose phenotype is generated that can be sdlected from a galactose negative background. HFT lysates, permit the detection of galactose negative segregants from transductionsg clones derived from galactose positive recipant cells. Traneductions in this sense have facilitated further studies of the interaction of the galactose loci with the Lp locus, “8 The relationship of lysogenization to transduction | By exposing cultures of Lp” celle to HIT lysates, diluting, and then plating on galactose medium to obtain isolated colonies it is possible to study the behavior of individual cells with regard to suet transduction and lysogenization activities. Table if shows the results of an experiment in which 1.1 percent of a cell population was transformed after exposure to a HFT lysate. The second portion of table 14 gives the phage reactions of the galactose positive (transductions) and galactose negative colonies derived from cells exposed to the HIT lysate. All of the transductions were lysogenised or converted to the Lpe state while the non-transformed colonies were either phage sensitive or 4 contaminated with phage. carries These results suggest that lambda f@ the transducing activity. could be argued However, under the experimental conditions employed it ia=pesebbte that the transductions are the resultz of the action of two entities. The would first, which,actg upon the cells and makes them "potential" transductions, and the second, lambda, which in the process of lysogenizing the cells, would sometimes > opi & , so many phage tontacts to res uit m convert@ them to actual transductions. In order for transduction} +o-he—— ° ? A ( V3 Q 3%) obeenrcd=at- | hypothesis, the "potentiating" agent would have preseat “th about —— -: Overs . to be tie-opder-of ten-fold te excess ef, lanbaa./' Et might be-argwed that because Gir kukke theoxperinent sarecorted—in—tarvke=24) onty-ubeet—ere- thist of=mthe—leambaameell—eentecte=became t rerniuctions=thet the ratio - of the "petentiatingll_sgemt -to iembia was wet bth. ebe-would-not—necessarity pe meré = ae aud w hick culfures we ve mee! “ slow posibwe | , trul wees “" celecked, Tet galachse porthwe Nansduchms are wel reads ly oe DI s6USSION The xaux results presented above can be placed in an orderly fashion by the following scheme. When lysogenic cells are exposed to ultraviolet radiation and the prophage is induced to form mature phage, on rare cccasions a fragment of the bacterial chromosome is included within a phage particle. When thie particle injects its genetic material into another bacterial cell, the fragment is also injected and if the recipient bacterial cell has the proper genetic constitution the presence + of thie extra genic material is made obvious. The—frapnent—renains—within ‘ + The allotypic fragment usually persists at cell ditision, so that segregating clones can be maintained indefinitelyyin mass culture. At least two additional events are inferred: (1) diploid crossing over leading to reorganized digenotes. Since these may be hetepogenic or homogenic, a Four oper sea strand (or more) stage is implied. (2) seergetion eccurs leading to ctieter ne heplogencte, the state typical of E. coli. The fate offthe fragment is unknown, Crossover haplogenotes (amphitypes) have also been isolated and may represent either a third process, or the first two in sequence ( ef Pontecorvo, 1954). Since heterogenotes give HFT lysates, the fragment or a replica of it, is assumed to have a high probability of incorporation in the phage obtained by UV induction. The low yields suggest a burst of one phage particle, a reversal of transduction. | From this description it is evident that the genetic transfer is intimately associated with the process of lysogenization and lysogenicity. Concerning the process of lysogenization in K-12 little is known beyond the fact that cell and phage interact, there is a period of indecision, amd the Oa infected bacterium either dies or generates a clone containing lysogenized cells. Once lysogenicity is established the capacity to produce phage behaves as a nulcear gene that is closely linked with a number of loci controlling galactose fermentation. - The rire step in the scheme is the inclusion of a fragment af sum within a phage particle. In Salmonella the fragment is a random , section of the cell's genetic material, but in E. coli K-12, it is m quite specific, for only a restricted group of loci are transduced by lambda. Again in contrast to Salmonella, "lytic" lambda is incompetai/widt in transduction. This may reflect an inherent difference between lytic and UV induced phage. In the establishment of lysogenicity the genetic material of lambda enters the cell and adsociates itself in some way with a specific region of the bacterial genome, In the induction process it is presumably ‘emerges from its place and starts to mitiply. Transduction could be gccounted for by some latitude in the separation of the galactose loci from the prophage linked to them, and their common bnelusion in some nature phage particles. The close genetic proximity of the galactose loci would suggest their increased liklihood of gnclusion, but there is no closely necessity makquiness§ genes be also spatially close to one another. : CBT wa VET) ‘There are two ‘types,of culture in which » tranpductng particles , ere formed and it ts Legitinate to ask whether the two are ‘different phenonens « or tex nerely quanti tetvely, disterent aspects: of a a single phenomenon. The evidence for a unitary process is negative in patugg. tat ies. no difference have voen noted vetween HFT lysates and art lysates, . YF he Lalita ated vee hens except possibly the higher incidence of tradetustions, “ath, Lp", react . with the former, This exception, if it be one, “gould itself de oxplpines on ‘the basis of quantitative differences between the two lysates. of tue = ent ta, of transducing particles in cultures giving | tr Saysates has not passed beyond the Preliminary stage. The evidence thus > mest et why far suggests that ee PTE of the cells yleld transducing particles The defeveunahin of “With ant that the yield per eel), ial pot large. wa regard to athe frequency of cells oat tting transducing activity it should be noted that cul tures _ , started fron a single colony with HP! property may contain #8 much as hie Se Sacgshm 30 percent of cells wlth MFT proper ty “* Mipeaees| gehe aes a” Doge The Saataeme of ‘the lysates: of segregating hgerosygous is @Alactose posit2ve clones indicates that the fragnent preferentially fg Lpoluded within the phage particles, oo - Presumably exchange between = Cadalse’*"™ gee fragnent ant intact chromo sone joscury mah that instead of giving lysates predominately allotypic in character, idiotypic lysates are obtained, The exchange ia sufficinetly rare, however, that observation remains cbjeative in nature. The nature of the association of the fragment with the infective phage particle is not known. Presumably the material ts within the phage membrane since it is not attacked by desoxyribomuclease. The availability - of lysates in which she most of the phage pabticles have activity (HFT lysates) or have no activity (NFP lysates) suggests that morphological comparisons might possibly be made via electron micrsocopyof intact BEAES PRELTGI“S or disrupted phage particles. The fragment enters the bacterial cell in company with the prophage, by analogy with 12, probably by the injection process (Hershey and Chase,1952). The association of the fragment with the prophage in transduction to lysogenic cells cannot be stated in the absence of phage markers, since it is not possible to distinguish between the previously carried and the newly enstered prophage. The carriage of more than a single prophage by cells of E, coli K-12 has been reported by Appleyard (1954) and it is likely that the transductions of lysogenic recipient cells are also carrying more than a single prophage. In only one instance, from more than 250 segregations studied, has segregation from a transduction of lysogenic cell resulted in a change at Lp. In this case an idiotypic segregant became Lp®, and this might have been a spontaneous "mutation", . In the transductions to Lp® recipient cells the associatéon between transduckng prophage and fregnent is possibly better seen, These transductions are of two kinds, Ip” and Lp’. All/segregants from Lp* clones have been lysogenic. On the other hand, Lp" transduction clones segregate Ip" /Lp* as well as Galt/Gal-. The incidence of Lp® Gal- idiotypes supports the notion that these loci are linked. In considering the rekationship of the fragment to the rest of the’ genome no specific statements can be made with regard to its perpetuity in the heterogenic clone. One would depend upon its possession of a functional centromere, so that it would behave as a small autonomous chromosome, or the fragment would be attached to the homologous chromosome attachment segment, either intersitially or terminally. Either, position presents difficulties for crossing over, and the fragment as a separate chromosome #,.20ems more plausible. | . In the above sections the results have been treated and discussed ‘dn ogeneral way. It is obvious that the study of this transduction system has only begun and that many experiments and intereéting observations will be made before the problem is completely understood, It is proposed to investi~ gate lambda transduction further along the following lines. 1. ‘Whether the production of transducing activity in aFT cultures is related to the interaction of radiation and cells, or is the result of a matation\ Like event in the cell poputation. 2. The production of transducing particles in HF? lysates. 3. The action of radiation on transducing particles and the possibility of inducing mtations. 2 @ 6. Farther studies on crossing over between fragment and idiotypic > loci using additional markers. 7, The relationship between lysogentsation and transduction, ank between lysogenization and crossing over. 8. Estimation of the gene order of the transduced loci and their xet relationship to other mapped loci, 9. Study of the biochemical steps controlled by the various loci gud the fexxeuxtzi fermentation of galactose. 4. The detection of other loci within the transduced region. Syngam ae 5. fhe bahavior of the fragment transduced during | meiosis, SUMMARY A cluster of loci in Escherichia coli K-12 was found previously to control ‘the fermentathon of galactose. Lyeogeni city for the temperate bacteriophage, lambda, was also found to be closely linked to these loci in crosses. The phage lamkda now has been fqund to transduce these loci, as can be readily demonstrated by mixing lysates of galactose positive cultures with galactose negative cells on a selective medium, EMB galactose agar. / The transductions result in clones that are heberogenic, that is, they are diploid for a small region of chromosome. The small fragment of chromosome transduced appears to have a functional centromere, and is perpetuated within the clone even after many single colony isolations, but it may on some occasions be lost. While in the cline #t has been found to eresnorer with its hoftologous region, On some Occasions at least, at a four strand stage. Each of the new phage particles formed in lysates of hetergenotes has a high probability og containing set—owky—o-deeguent, wet the fragment iemth.tee—casmemey carried in the heterogenic clone. A position effect on the expression of two of the transduced loci has been observed. Dtheterogenotes of Gal, and Gal, are not phenotypically galactose positive in the trans positiong, but are so,in the cis. 4d ese BIBLIOGRAPHY Alexander, H. E., and G, Leidy 1951 Determination of inherited traits of H. influenzae by desoxyribonucleic actd fractions isolated from type specific cells J. Exp. Med. 93, 345-359 Alexander, H. E. and Redman 1453 ‘ Chante ta , ty af Mening 9c? ch, t . Therionc ryge weaucl of Ce peafe eniPacks canteoning desonriboruclel ve - qed. fh test ved ag 1941- wot Eppleyard, R. K. 1954 Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from Escherichia coli K-12 Genetics 39, 440-453 Atchley, W. A. 1951 Cold Spring Harbor Symp. Quant. Biol, XVI, 441 (Discussion of Lederberg et al) Austrian, BR. 1952 Bacterial transformation reactions Bact. Rev. 16, 31-50 Boivin, A. 1947 Direcged mutation in colon bacilli by an inducing principle of desoxyribonucleic nature: its meaning for the general biocemistry SEXRBERALEZ of heredity Cold Spring Harbor Symp. Quant. Biol. XII, 7-17 Herehey, A. D. and M. Chase 1952 ‘ Independent functions of viral protein and nucleic acid in growth of bacteriophage J. Gen. Physiol. 36, 39-56 Hotchkiss, R. D. 1954 Double marker transformations as evidence of linked factors in desoxyribonucleate transforming agents Proce, Nat. Acad. Sci. 40, 55-60 Lederberg, E. 1950 Genetic. control, of mutability in the bacterium Escherichia coli Ph. D. ShakSmniversity of Wisconsin, Madison, Wisconsin hederbers, €,. and J, Lederberg 1953 Genetic studies of lysogebicity in Escherichia coli Genetics 38, 51-64 Lederberg, J. 1947 Genétit-recembination in Escherichia coli Ph, D. Dissertation, Yale University, New Haven, Conn. beevber J, 1950 g! Isolation and characterization of biochemical mutants of bacteria Methods in Medical Research 3, 5-22 The Year Book Publishers,Inc, Chicago, Ill. Lederberg, Z., E.M.Lederberg, N.D.Zinder and E.R.Lively 1951 Recombinational analysis of bacterial herédity Cold Spring Harbor Symp. Quant. Biol. XVI, 413-443 Lederberg, J. and E, L. Tatum 1953 Sex in bacteria: genetic studies 1945-1952 Science 118, 169-175 Lederberg, J. 1954 Recombinational ,mechanismse in bacteria J. Cell, Comp, Physiol. Supplement 1954 ( Symposium on genetic recombination, ORNL, April, 1954 Pontecorvo, B, 1955 ; - Analysis of mipbtic vecambination ih Aspergillus niger ‘J. Genetics 52, 226-237 Stocker, B.A.D.,cMsD.Zinder and J. Lederberg 1953 Transduction of flagellar chafachers in Salmonella J. Gen. Microb. 9, 410-433 Tatum, E. L. and J. Lederberg 1947 Gene recombination in the bacterium Escherichia coli J. Bact. 53, 673-684 Weigle, J.J. and M. Delbrick 1951 Mutual exclusion between an infecting phage and a carried phage J. Bact. 62, 301-318 Zinder, N.D. and J. Lederberg 1952 Genetic exchange in Salmonella J. Bact. G+, 679-699 Table 16 The transmission of heterogenicity in crosses parental elle ; Prototrophic recombinante Zz ¥F G + & - Galo=(}) Galy- tp* 1" abcut 6000. Galye ip’ al,=(2) sue 99 * unstable for galactose fermentation, 6 galactose negative segregants tested were Gal ~ ** 25 of 30 examined were unstable for galactose fermentation. One segregant from each of the 25 was tested, all were Galo~. (1) contrel platings showed the ratiox of (+)/(~-) in this culture was 109/57 (2) control platings showed the ratio of (+)/({-) in this culture was 115/13 25 Gok” bp® GoLt Let 324 } 757 (aprox) Gar t te Gol. ig? 107 (ppror.) Table 1 Principal cultures Wisconsin Stock Number Ge mtype* W518 rou Lac,- Gal,- Lp® + + W750 FPF M- Lac,- Galj- Lp W811 FY Lac,- Gal,- Lp’ + w902 F- T-I-B)~ Hal,- Gal,- Lp + W1210 F M- Lac,~ Gal,- Lp’ W1436 F’ 2-L-B,- Lac,- Gal,~ Lp* s° W1924 FT Mu Lac,- Gal,- Lp™ W2175 Fr’ Gal,- Lp* W2279 F* M-Lac,~ Gal,- Lp® + W2281 F M- Lac,~ Gal,- Lp® *Genotypic symbols reger to the following characters, (1) Compatibility status, F (2) Nutritional requirements; M, methionine; T, threonine; L, leucine; By» thiamin {3) Fermentation reactions; Lac~, lactose negative; Gal-, galactose negative; Mal-, maltose negative (4) Phage reaction; Lp”, lambda sensitive; Lp’, lambda lysogenic; Lp, lambda resistant, but not overtly lysogenic. (5) Drug resistance; S, streptomycin Table 2 Recombination between the various ig? Galactose loci Ww wo Minimum Number of Percent Galactose Gross Prototrophic Recombinants Fermenting Recombinants FiGal,- X F Gal,- (1) 1500 0.13 (2) 6517 0.06 * (3) 3603 0.03 “ 11620 0.06 — ¥’ Gal,- xX ¥ Gal,- 4588 0.13 ¥ FY Galy- xX F Gal,- 2654 0.23 FY Gat, = W750 . E- Gx2, - W150 (aevalhm phenocopy) Fo God,- Wier Ft Gely” = weil wsig wit3e Table 3 on /48) \ Observations on lambda lysate transductions Locus Number of experiments 1. Leci not transduced Lacy (serine or glycine) Leucine Methionine \ we y Streptomzcin Xylose Proline 4 Ww r- WwW ~ ro 9 l(lytic lambda) 2 1L Cultures involved w112 W1678 W1736 ,W1436" 58-161 ,W811 ,W1821 W518” wi821° w51st W1692,W1920 ,w2062° 42062" W2331 ,W23478 W2071 23079 2, Loci transduced Gal, { We ate As Gal, ( gs . Gal, Gal¢ (Footnotes table 3 continued) f- lytté’ lambda grown on M~ culture g- lysate of prototrophic HFT Gal h- lysate of prototrophic HF? Gal 2. 2 culture culture W750, W2279 ,W2280 ,W2373 W1210 ,W2175 ,W2281 W2297 W518, W811 ,W1821 ,W1436 ,W1924 w2070 oe ey Ww) Table 4 The interaction of lysates ani cells of falactose negative cultures Recipient has ‘ay Galy- Gal,~ Wild ‘as pH aes Bet 2 : hel Art Gal fr (2) e196 43 - (2) 2 2 - - 40S Galo- = (1) Us 52 lL 43 - (2) 20 - 10 - 356 Gal,- (1) 89 - 202 - - (3) 59 85 - - 417 (3) 4? - - 30 394 *“ The no added lysate plate which represents the number of spontaneous reversions occuring on the plate. The remaining figures are the mmbers of papillae occuring on the plates per 0.1 ml of lysate , added, Table 5 Restoration by reverse mation of the ability So transduce previously non-transducible lock celle (int Reversilo lone uyeate Reversion Gai, M4 = Gal, (1) 0 eves Gala~ Gal,” (2) 10 96 Gals” (2) 6 552 Gal,,- Gat,” (5) 39 204 Gal,” (8) ' 25 291 *number of papillae per plate, 0.1 ml of lysate pleted, Table 6 The necessity of of lambda adsorption for transduction n¢ bbe? a Plate, Addubun- Recipient | ’ i Oipptiee Cells (L ___ Hone Wild type Lu Ses Gal)- 8 1 426" r | 1 2 Galp- 4 20 356 r Ww 14 Gal,,- 8 / 89 296 ¥ «50 57 *xumber of papillae per plate, 0.1 ml of lysate plated at sc ROned-+- Darmeabet | nr fearbda -2 Nroltict. pone dt wt dane atu lok da oy Aomubde -¥ _ Bable fe The actiln'ot Ivtteally aom lambda, Plabe obbctur Experiment Recipient Lp Rial: cells Allele ___ None Lytic lambda Place titer 228 Gal,- + 3 2" 2.4 x 1020 Galo~ + 9 8 Gal, - 8 9 8 239 Gal, + 2 o 2.4 x 10°C Galy- + 6 2 Gal,- 8 13 8 254 Gal,- 8 - 6e* 2.4 x 10°4 Gal,~ + - Bae Gal,- 8 - | ore Galy- 8 - 6 Gal), + - 39% 280 © Gal,- + 0 Que 6 X 107 Gals- + 1 266 Gal,- + 14 10** *Papillae per plate, o.1 ml lysate plated. Lysate prepared by growing Gal,~ lambda (UV inducticn) on a gelactose fermenting culture. ®*Thege papillae picked and streaked on EB galactose medium and found stable for galactose fermentation, Table 8 The specific activity of lysates of the transduction clones Recipient Transéucing Titers + Cell lysate fransductions on Lp assay cells P/T* Plaques Gal,- Galo- Gal,,- Gal, - wild type 5.8 x 108 2.4x106 1.8x 107 1.3 x 107 32 Gal,- Gal,~ 7.22109 1.2x10° 1.0 x 108 - 60 Gal,- Gal ** 2? x10° 1.8x10° 63x10 = - Gal ,- Gal,- 6.2.x 108 4.3x107 1.5 x 108 - 4D Gal,,- Gal;- 11.5 x108 5.0x107 7.5 x10° 74x10? 8 2 Galy- Gal,- 9 7.9 x 10? 2.5210? 2,8 x 10° “ 29 * Ratio of plaques to transductions, the maximum transductign titer observed is used for this estimate. Usual ratio P/f is about 10 - *® A second isolation, The occurrence of q Table TS stable transductions Recipisnt Lys2tea cells mfg Wild type Galy- Gal - Gal =_ ceo * o/c 0 7 PC Cc if %8C C TF Galj-Ip® 38. 2 34 - = = U/l 2 NL 30/2 1 29 Lp’ 46/1 1 2 - - = S/1 1 4 27/1212 27 Lp* 143/1 1 42 - - - 9/1 1 0 - = = Galo- Lp® 46/0 0 15 214/00 27 = = = 98/0 0 & upt 208/17 17 21 83/4414 61 - == 79/1 4 52 Ip’ 23/4 4 6 65/2 2 0 - = = S65 5 0 Bal,- Lp* 835/19 19 383 «= 72/29 29 72 492/11 11 20 - - - Lp? 573/41 42133 (96/51. 5196 ee - -- Lp” 320/31 31 127 - = = 238/31 31 50 ~ - - * Papillae transduction plate/ papillae control plate. T = transduction plate, 0 = control plate ** Corrected for sample taken, stable obs. X Papillae transd, plate galactose fermenting papillae. sample size’ With the exception of the T/C column, numbers given are number of stable 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, (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. 928 19, Loss r ad al Peapics tat Bune tes L355 v MATRIX TO TEST TRANSDUCTION MAP SEQUENCES G2) Secqmence Codes for multiple exchange types Goer VOLE (Operators on donor gono type ) 2upoint test 123 b- 132 c 213 a hepoint test 123h b e ac be bd 123 b d ad bd be 132) c b ab be cd 13h2 c d ad ed be 1423 a bo ab bd cd The complete table can be 1432 d e ac ed bd generated as the permitae tions of (ath, cd’) where — SL3h | c be ac ad atb=bb, be, od, and bb=b, Zu) a a be ad ac . 231 c a ab ac ed 233 d a ab ad ed 312h a b be ab ad 3201h b a ac ab ba Tnstzuctions: %. Write dew the donor genotype (differentiel markers only) in any arbitrary sequence, Sales Wee at Y+ Ze . 2, Group the experimental results into the rare and frequent classeg. 3, Cede these classes as transformations of the donor genotype. The code "a" moons "reverse the sien of the first locus written", "bo" the same for the seccnd, etc. Thus, (ad) (WeX+¥4+Z~) would be W+X+¥+Z+. h, The table gives the codes for the multiple exchange classes (mec) corresponding to each sequence. Those models are excluded where frequently fourd types are included in the mec cedes, and vice versa. 5, Tha sequence codas can be translated into maps by writing the donor genotype 4s WX YZ and transposing accordingly, Thus, 231) would be the map XYWZ. L23k Yor the reciprocal transduction, superimpose the operation abcd, so that, Ccefe, ac becomes bds c becomes abd in the mec codes. J. Lederberg sf nw? WET. eBomyee’ Mr aed obonygt: CP Tha to Some Teper e: Veh ae Rpt Arh rarer a Reel Guba untth Nowaite ANE a oh bai = rege” ET Al nt flees - bee 4 Mp. Get Eek ko. Slh ue ee Be foe . _NaWnng WET NET, Maca ee | Spetantines Ge. co ee we (oe “ Avesh. wit i on FO Nn Mee - Corman bs god se fe Aa ae Pela HET hg obdny Magee Ure - ve (a2 - fly wid! NET Let “pubachdeg 2 nent pene 4, (270 2 LI Ls : yo. 206 +o, : jt u a - ie, Me 39] _ de _2t3, 21] 3¥0 f FADE he 273 4W 279 377 - t aA Ep nee bet faye 4G 337. Bor 219 —8tIa ust 4/3 3 ! / 7 2F0 a 230 a4 gy | 0 xt ® 1, 298,295, 541 | 270 271,28 r tt LM, S357 31 ) _ hb =* a toe 7e co [7 —xb- [2 =x 27 — Found ms e's. . . _ Prin pet ewe 4 6! 5. 5 | 6 223, 4) 2 ¥, 257, 2% 268,216. ont, WL 2 Bor, 9°7 ,3i7 - 3 S07 307A _ aad pons . - - 330 a AR AL EF BAB BH. 34u 33% 33 24 eed wet od. ae 3 247,37 BSF ts 23177, 1P} Soa, Bob, Bf {3% poet Su? , 357 : NE ei ee e or _ #7 XIU - ; reo ty 8” grax I FT Vw “-—K 2- oT x FT + lng 2216 ae Loa a ULIT Vb py, 24]. afb, 32# 333, 40 217, +e Bo37 227A, 233 23CA 247A Lae yt (1360 | By 2¢7¢ rb Grr 221" ¢ 1 1337 cee ee fee Lie 249D a 348 9h 903 agi LOS 9% 33) . Joy. aie 2 ar . | dhr 287, Le 24 IE. . : bay " me Gat ALE ate K (by bau Geten (9p- KGa ery OK Gor t t Nee bux sles oe ty Coty x thers - (AHL LIN H | eh. dae pant _ WW. Her Do Vedas 4 nm (ne - ben Gat I Che yuk baal, _— (beGeone, po x Ot 2347 aef 277 WES fo, _ ST _ thl rar r9e 239, UE W227 UK B81, SF ee 297 : | UE LUT, UB) wwe 2 253 rS, 337 335 HL, 49 U7 ML 322 223 32 } ory 3s) BSA 3S Gay FY KR OF ET od Meee FO 335,437, Guty7 G4! ~ Geer Ugh _ _ — ooo ene a _ ee . . occ ee ee ck IE OT ee FL . cee a ae : 3iy oo 7 + —« 77 33-0 ; . 4 x ro- 32u yee wee Sop - GC ExT” - _ 323, 399 34% ee 323 JuoKIT $v? AT eI 6 | 336 Ail 2 x rb ue? a . Ce PH fet 77 GK ae 3] i 246, oe At oe 8 G7 ra Shy o 4 lw o | - 77 GQ 7/1 s o 2 oY 53 SpA Id. Bez 1 37523 / e tt DY [eee if (325-7 Haney iGO izo | . 3275-44 nl oO 34 “pe tgs) a ge MR fo | 7 | eee tO a — - . a ee ee 47 | oO _ ‘Leo & con 37 iS 6 -- $7 6 st -~ a ae + oye aay + po ; 3bh1 = 4 be pb sa /s Ss gg Os S Fo SH] Cade Ele bag pe E. + a os Wye Ir Whi 3 Bhr ¢ is. 3 ——— 51/83 (263) 24 0 0 oO . o . 3.0 “Bob po 4 sf tbe 6 It x 3 pe qo. ‘S: b — ~ Wt 10. thy 4+ + if _&fe0 2 Fe? 3¢0.2- pF : q> Z- BUb-2 Fo 47 sl OK ‘yo ed ER hy WM SS oon - wiles) a a ee aa ‘ - a- wat ia Je Peng hae iy a 6 2¢Ie-4 git {arly “_( A tusie lll) 9a ——— QR er re mre ee 3 an lp® (3A pertain Lables), , . mer Veneta ee 4 Ton lp SAC preowss. whee) 242-2“ 7 — UL ttt Jing — 802 | Ngy ee ee A tot Abend apts gen —— __ 7 ? Bee at Lett ter shat hae 6 - yt K Gtr Auitasin SK 3 _ 234{_ ihe 5 Lh. = _ age by nena Vy cceuroels WINE vat be pint Olle VL net den Uk a : re oe pot ia ae | atte _- ites Oven hn DEL us tue. States 800 - oh a. _ er. vl howd’ 2¢l-¥ a BROTH MP a ee ___ 309 a HOT, Got Km Gea” Gt Heaty = 2380 X— Gay- (ner) a oes > _ Gta tye Gre. Oy Ber ae Ah = pert wis lend th Cat gy ee oe ee, 0 eS oe ee bf, 3 wer “ “ oo ; Ye gat « QO, #* a a Seo (ip 7:0) . o\se Wee Cry te be bane d. . reas de wt ee AT ar efit 7a J | : ao _ 0 2¥/ — | d- a 8/340) 3 Ww | SK t-te } oe | EZ ete ah nab ast a Les Ro- io eee —_— on yes re F- 6 Bfagye g- g- 443.38 (32¢-), ———> 9/j90 (ue) V7 4 Basie (7) 4 OEY Ge) Sibi (Boe) Cp Ee nal va bet Qe 4 UO i/ 34 Yo! 8 t+ 2 a Eg . + 7 ; j teeathnakl fmm os q- | OO Gt. 2. + 4 s§97 e _8 me id 4) _ _ _ ce Yo Q Vv . Thos edt Oh temp _ —__ _ = —Be ) aon _ Va o,- oifean vais (10) QC 9 Cray dow (Ju4, j_f1o)_Dixaer (172, VTY)_ Dy cece He (074 (174). DV a wer 24 Sunes is). DI-K AE 402, row poh smae( V0) D1 the (et) 8 tre dawhen DK Ges) ware (ish) DIL @® Gor, d4%, 143 Cie) lo KD) 1D (a) oo . 175, she Di Ale3 WANG | (Ih se3,) POERBUOPYAWAMM NESS) DIKES Ose) Mo ter, sung bu (Car LEED 7 Got om (140) i clay (2.6 wats) (143) O1 vs Goch'tovune C14 iM va) (3 | a Cha (A) fos," Cugae) Nt Gane. ber. (ar) neste 9) (Ou, acct af) (1are) LSet wie enc lt) . a He turns) spua(Oeh wart me Vs; fz ; : Gum (ius) Vx aor (ae a4). ot Gus’ (128) vt-xat net (ey —{-F sare cay (1428) Ue soe HEF (14D) 2X Cor, SP ((498)_ WIKIS HA) (rehome sever (se) _ _ Ss Gea g* sht (ed) ; _. ae oe _! NiG__ (e At SS NNO" parse] ) Gat at Gab) wre ger (Ste) (vn) NIG 9 LET Seq GS sete (iqre) NtO Ler vey (car? (142 ) wie. nese) Rt TS aon Ne Ke (oxide) Csi). phx 23 > Cw ~ (wr¥32) (203) 5 ofan Het (24 ) vib—xi 1282 (2341) { ay) Vh Cu $e str ( 227) - — 6) BLS MWA is 3 (SIL) senate Gt, Qer) X14 3h 740% Cost) Lye aloe G40), tae der (24) | ‘Mee MV (Bev) Qu the Mote MS de un.( 0b) —W) (sit) anh boty (22) Ber [e,_ Cot, Coty” wile Gu May (21) en Sa Atse cor a_As ss | 2346 (_ _ 7 — 1 he OA 7 (NA) qor—«° 1 (232). Nat—x br (2 4) 2229) (249) te ceatote | ooh sWA (274) wo J in ja (ses a er One Ay Cata® yolk | (1) cfr ta cote, RA ft stk Gzs) L (ay bee* att auabain C44) vee Her (248) marly Cod (FATALE Gor) _weed Met A) open OE ee Core) ter wt fase B82) Her degctet BS oxi OM 19) “Gur—x 70, ou,- (41) | ocd. leaky Dr 276) Yn ars” uth f (2.70) LET any a (279) ) “eS bo” shh PATE) Su Fr su bed Gut~ Foy soetalabe, 247B-1) | UY T-r tn-x770 bes, - OA ara a &G- Gee fate (257 : ~~ {2774 dot 41 bot oe O59 ce elu Me [969) fa / _ eee ee vee —“AWET-04- pe Borne — tend. eS bth 2 peor — w eT C88) Lee ter Cr) aa (x) % te Ye bu hut waft tunitlt (Bor) % bey, sud Z eat ae. ir NM) ht ee uw shel fe (Zee) Gut shot hot (746.) 247 tre ? 4 CH che Hunihe (760) thd 2a) Saleen (QG3-Ik alu sa (2483) ) bed | 39) wer T Gust? ee — beat. (374) SFr any. (334) — EG acne Gi ne 09 nail, _ 34) _ tay, Gy ne _ Cx xa Yi x rene (53). Gea A (33%) 1) Gras" (va oy, _ Pr te 27 G1) on ——______. Ga Gana AMIN (943) tea G5) ead ) My oe wash tat sh (205), a ACS O~ UES AH a | 283-1 —xja10 | (21m) Y% cost* we wel Gaon —« 2307 1 (302) Att (24) ov Wametg fares) (6) mos ee “ye Geng ues hy (263 3) UW sad: Bes) Ler om 77 (340) - oft - eee (CIGD Dore\t —x DIY Gu) ie rel my (3B) Leta t- (1092) 9p oy (370). B 7 “Byl-4 alee (375°) th Ges wert oy. BeS= BU —KIShu or). Bes Saat eu. Pe Catt cnabe vn BG mew by keh Yo late omy 2 God (3tY) — —. \ —_ a lls ent _ or Sat — —— Preachan Colt ~ Fecha of Get ns Phtipe Derived bi: Revevisoms Sequege true Phos hye Raversuns Seq reqs bus : ; a Gee = Wh rts hae 7 | - ee: or | - > ue - ° _ . : of, VY wid Yr __ Ae,- &/s i A Get gS aytyate ee G@t- 9 7 es —_ oy age | _ a 3 oes ge Wy ; oo : (rs, _ 4/e ~ - Pe Cae Rane Solan r/, . . Gat 1 ‘ O/, Mie Gt Gtr went thlted a | ee of, rn TE “oh (oti) M8 ee (F) - - - 7 ° A (m™ n toe ~) VL at - ~ ~ Gaty- ° A (otra) (1) 5 . . _ : _ OQ 4% - Qe (+) fy (9. /4 ow Get, Haw oUt - fy, ce, - i xa [x 7+ % ble balye srt /iteye fable 5 The frequency of transductiong unstable for galactose fermentation fella dal (+) Gaye Glen Gals Gal,- Lp* Yg/22(41) = 0/11(0) 0/29{0) Lp* (1) 23/24(96) = 23/24(96) o/27(oy Lp*(2) 17/24(71) - 24/ 24(100) - Galo- Lp® 28/48(58) 63/72(88) - 64/72(89) Lp*(1) 22/24(92) 19/24(79) = 16/2!( 67) Lp* (2) 16/24(67) 21/24(88) = - 22/24(92) Gal)- Lp? 13/24(54) 0/-72(0) 21/24(88) - Lp* 20/24(83) 0f96(0) 19/24(79) - Lp™ 29/4%48( 60) - 18/24(67) - The figures shown are the fraction of cultures unstable for galactose fermentation. Percentages are shown in parenthesis. — Lie. HET valet | Gis bet, SE GS | ulin phen. oud Motha - te poten Peta pred by. OC fan wn a Anarene , wad. _ a by ke ee dt. 0). — een aalile renphne fates, he poo bee Poy deep & “teen Vpee ee pinniane Sematt D, 49 I) on Oe fern Areas to waed Lat. pds cote Me Lehn oe Cobh ( WW) - "Ty ee 9 a mtn? ond Ke ___2gre bert Mapatrer Tur hiro NOws Cre ho Sur, Aye | be A perp . , eee ee Cis) “Tr ha. Annem Mey. + - pte Ute Te. So ~— bec (Me) “The. pete (oh 4 rence co Cet OI) ele. wee ee Aen naa a i | Sha tet 4p sTuasQee94) Lees - RRA Seon. TRE Ate om lee hr HW a AN mse a cee Sem pee adil fern oO Qq Lece wee " dann (168). Pe oe. feo. et. an eT fee | CAM nee wR lean a %& Aen. | Tobie — | : Seger Behe 4 Perr ixs t (Sy, y? Gund by & Segoe . Ne. Uns Toto Hate Set is se Fuad Saag, ee ——bpE de - Heo 2 a | 8. , Gty- ip’ I . _ _ “Helge ; | ; O 2. [0 b | oe Oe Weta i“, 287 re pie bo, + Let — oo | oe ir cs Sl Go, | : Vir, vA OS st Ody. GS Gyn Lye cece eee cee ce ren ne ete ante ee ee 302, Grae, Feet byt / [Gd bok) ee : . : SG a tha US, 7 bate yur we DAG YAN wpe Tae es uta oo “ | (YY Cis-trans position effects in transduction heterogenctes of ‘Hecherichta coli — The phige Lambda ¢ oan . transduce a fragnent which includes a cluster of genes ‘for galactose fermentation. Most of the transformed clones are "diploid" or “heterogenotic for the transduced genes. Many combinations of non~allelic Or re ren mutants give galactose positive heberogenctes as readily. aeGal+/Gal-. Howes wee ee some combinations of @al- gave smaller and delayed. yields of Positive. clones. > ustion from Gal, + dal,+ Further analysis disclosed a cis-trans position thet between ooragt loci. Tor example, while the chs +/— heterogenctes fomed. wy to Gal,-Gal,,~ are positive, the trans ie heterogenotes: trom the trancduotion from Gal, —Gal,+ to Gal, +Gal,,- are phenotypically galactose negative. In the . _ negative clones, positive heterogenctes are later formed “py orossing over in the, =ty Mt, and ==. The delayed yields that were. womens tmatetty are tuahon _ these secondary events, sh Reciprocal ¢ transiuations have given identical — - ‘phenotypes, 30 that in netarogenotes the ‘genes in the fragment ara functionally equivalent to tne nondlvegous genes in the chromosome. The galactose positive "phenotype thus requires that + alleles be in adjacent positions either in the = fragment cithe chromosome. - PaPiLLAE PER PLATE 3000 2000 | 000 LL {he \, X t ‘ A Mag Gal Lp* Galg” Lp* — arama ‘Gale Lp” L 2. 2 3 10" Lambda Particles Plated PLATE PAPILLAE PER 1000 500 Lp* Gol” Mi. of HFT Lysate Prareo ‘Vv'S ‘nN NI 30vW ‘OS NAOZ13IG0 AN3BONS wsdvd YSlLawiaiiw Hdvyo N|AOZLaId W- Ire "ON \ ON ty 4, Transduction to Lp® recipients It has been stated previously that transductions to Lp® recipients cells with LFT lambda + rresultd in lysogeny of the clone. Nearly all of there lysogenizations are Lp , but EXHXB rarely a clone BE with Lp" phenotype results. With HFT lambda there is r a higher frequency of the Lp type, a result which may only be owing to the lower " armbybrn we chances of secodary infection,with, HFT lysates. Of 58 syngenotes isolated as sings colonies, 13 (22 percent) were of Lp” phenotype. *hese syngenotes were made with different lysates preparations, amictasre dérived from different homogenctes, and there is no indication, as yet, of an association of Lp” clone formation with either a locus or a lysate preparation. . . h flue Ald The Lp clones described previouskp are carriers of a"defective" prophage ( Appleyard, 1954), but EYES plaque-forming lambda, in small quantities, may be obtained from them after irradiation with ultraviolet. The Lp” clones obtained TX8mx with HFT lambda have not given lambda after UV treatment, and differ from previously r -t 8 descrided Lp cultures ip segregating for Lp, yielding Lp . Thus they appear to la 8 +. .fr 3 +,_8 be syngenoctes of the form al” Ip”_//Gal Ip. Segregation yielés Gal-lp', or Gal Lp \ r haploid segregants. No non-segregating Ip clones have been observed. This last observation suggests that the lambda "defect# in these cases is with lysogenization as well as with production of plaque-forming particles. / apap Obvious segregation at Lp was not observed when Gal+ segregated from Lp* recipients, and it was not possible with these syngenctes toc relate the function of the prophage to the genetic material. Lp _//tp* heterogemts permit study of this relationship. If the chromosomal fragment is independent of the Lp 8 genotype, Lp segregant cultures may be hemogenotic. Gal+ reversions of segre ants from Lp _//tp* syngenotes were examined for their segregational behavior. Under ; yr GA conditionis HXAX where the ES¥EH reversion’ test indicated 23/23 Ip’, segregants g ~ to have been honogenotes, 0/11 Lp , sekregants were found haploid(tabdle|0), the Although it is net possible to determina adequacy of the data, the indicatéon is A * that the Lp’ allele has a centromeric function, that Lp probably does not, and y x : + that the Lp® allele cannot so function. Failure to obtain sagresation of the Lp allele in transductions to Lp* recipients may only be an indication that the heterogenotes studied are not the primary product of lambda-sensitive cell interaction. a3, (424 ie Table |[° Segregat ignal behavior of Galt+ reversions of Ly and Lp segregants E-periment Segregant Number of Number reversions Phenotype Rumber Reversions per segregant found segregating 287 Gal, Lp” 1 8 8 Gal,” Lp> 1 6 0 292 Gal,” Lp" 12 | 1 12 Gal, Lp® 3 1 0 Gal, Lp* 1 1 0 2928 Sal” Lp” 1 2 2 Gal, Lp” 3 1 3 Gal Lp*- 5 i 0 298 Gal,” Ip” 5 2 10 323 Gal 6 Lp™ 1 6 1 Cal” Lp® 1 2 0 ~~, 8 +, Pr Hferogenotes. 2873 Gal, Lo S/Gal, Lp + 8 & + Yr 292, 292A ,298; Gal, Gal, Lp S/tel, Gal, Ip tu - Ss ~ + B ° G 323; Gal,” “al” IpS// Gal,“Gal, Lp YBLIWITTIW } , Yadvd HdVSO NBOZLAIG W-IPe ‘ON ¥ ‘YEN NI 3aWW ‘O29 NaOZLAIG AN3IINF IN UL B.A PRINTED EUGENE DIETZGEN CO. 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(rr. és . . bY ] 12499 | 1 X o-F | % vi 2h ¥ hed Oe DIE gSY fd (223 Yo | — G2) bq 12 (3) (0.08) el rt 2. t Lf f a 838 735x107 §.35x00" @ 5 0.602r/y bey W Fore Phptrsine A wheel . a \ aoe f- x (= Adin note peat oO. 1S fowl 4 ON ~ L- : a, 0. mk ju . ee bn 7 ae O38 her 22 yea” Oo SY 1 St 4. o as 6.3¥ (0.124) » 00 47 3ube% wevdt.- 0.34 - wens 10rd 3 / rol —tepem ee AM pot At Cutt Gut — TAA _ — ¥ = LL 344 6 ca PD ee . ; ~\ : az 43 a ¥ 744 beo % | _ - my . ; on { 374. 20 By key (2 cae oe [ & SL ; o le FD ed. 4.36 @.0%)- 0,030° 7. i ~ nn! ae ae Le _ 7 “SSK 7 Oo oO bo F ; ~ The pute rs “Because 1 _ “4 Dre selva, wea tak adn oat Clee ih Wp, Cus. $5 lant | $Ur-! yooh = hee tE [wink = orf Ny toth Get Gu bh bapa Pet | t 2 ° 6 ¥2 ~ ee | 5 HY oO. | au , —t/ “0 20 a ° L a a 3 5 ons € yerhy Papry pun 1740 ard seypIvy ) “_ . DINHIISVDOVINaS oa wanzasig aNaana BaeWd HdvNoD Nanz132IG Z1S1-Ore On + SOR ML Tore HOM! w3d SNOISIAG 21 x S304 © Defective Cale Segregants Heterogenote Number Lambda Sensitive Lambda Defective 292 2h 36 323 4 2 331 6 0 336 12 0 343 5 1 346 5 1 36 20 1 368 3 0 37h 9 0 382 5) 1 Misa 16 1 20 16 2 420A —2 wi Totals 143 h9 Meee 210) ont =e Gal; Ip* W750 Fraction Surviving le Sel Expt. 316 Procedure: 20 7.5 0.97° 1,12 172 Table 2 2/1/5u intreviolet, irradiation of HFT 2~, Iysate dihited 1-100 in XM), 0.1 m2. Sample removed and added to 10-ml, Pensasay. HFT 2~ stock = 2)3-1), nal~ derivative, Distance fron lamp, 50 om, UV Dose in Seconds Th aneraReeenittemmenramaprae ns s 2 B © ws we os 5 us am 10.5 17-3 22.7 21.8 23.4 2h.9 18.2 16.0 26.0 14.5 893 U3 63.8 714 53.6 os.g 79.1 69.9 71.7 58,9 6 28 39 Ww ww M5 3.00 O43 0.38 0.25 0.09 0,22 0.077 0.029 0.019 0.025 0,009 Aw ay No, of Trnsd, Tested No, Seg, % Seg. LpS Lp* Lp2 Bs Gene Types of Non-Segregating Gal+ ~ 7 Ip* ipt % 1p [oO oO 0 = Table 2 Analysis of Transduction on Gal, “Lp® Recipient 33 Ip Gene Types of Segregating Gal+ 6 UV Dose in Seconds rc arti CON AS 2 bs 18 18 1 0 6.0 0 1 = 1, 17 ) 0 2 1 88 9; 60 18 9 B 90 18 18 1 6 R R 4 17 i 0 0 0 9h 100 120 to 180 18 0 0 ‘VCS Oo ONI JaWW "O09 N39Z13IG 3N39N3F HON! 43d SNOISIAID ZI xk S3ngAD TIWHLIAVOOI-IWSS H3advd HdVYS) NAQZ13IO SiISI-ore 0 ‘ON tes Mima UV Dose in Lg sate Aebeid Dio) /-100 —~9 Olmwe Sangin fs bel Ca 10 tyne (2229 (789) | Pepe He - Ltelid HET Sait (attdnet-) FG HRP oat 2.7 ER bY dG “y N ~~ 4 ~f. oS ate NY > ~ Ww Cee ek a rate - joys 1K" 1330 SEE sR ~ ~ yor jes) SO A 8 rr 7 re - J 75% (x00 | 2) 2a 494 Sr HY P07 / 2307 [-30 ss ¢ -2 —2 a 3 3 ee eee eee TD | Ba BS BAT SSG SIE ~ 3d . Vipin /rsto txt \ 74 188 347 ser FATS 9™ 3¢/ au? ) 3s 23 _ _ Jest) ixetfas weip Hyg hw ARE Le ; eT OF 17 C3e ap - SCOK SO04 set Yt 234% le 7 | om Tb (009 16.4 ne Gy ar — - , i ie P9892 9 2s -no [he pheots 4. vt 4 Sens-ns| Is aslo ns 25” Lls-ths Uf5- as cs fe hate hero Ft spt fo to |r-*! 0 7 dee 1d weve oe Oo . repbeekm 8 pb. he 9 las vlad fed ahisy lar SiS, jr 74 Ay, ™\ f50 oF ° ee 1 TT | | WT rth . oth sptpent | estar lal= l= b- [=| (ANS | vee | oe [a8 [an | — @ 3 47 Os ° 39,7 3e6& FIS,, a7] 6 — pow is| 49s 77 & 2Pr Gr, rg ae “he f oe Tk ae B59- BSA -3SIB Jf Picke —f fot Eudo | 3 | i | 1 \ { ' ! 1 | i | i | | \ ' i | | ! ! i I i | : } i ‘ i + | weg gee 4 A dip Ufo o a _Netun ‘~ =? | | oar « nng A 3 moe 42 {he _It __._ 109 byt ate” | Evpt . 3 "oy [sf Proce duste . Uthanudet trroducghins of er ~ Cysake du luted [100 we D(m) Ord us S aang les re mee d Gud Added tO [Ome Pern ossee. HET 2- stele = att -id : Wol— ey ivahx . Diskiee Aw, laup , SO cur. “ash UU Dese iw. Secasdy Jyasa. . los~ 9350 AMEE! ° 5 30 0 60 7S 90 3m FF EB ee Be IFO Kt02 08 TO TT me Gol Lp> a SS. 7 OK ITE ART ahh 2H BAF By MO M0 TAS W224 GDL p67 rho 18¥ 29.9 192.4 ORR Hj S36 WE 729 O17 UT SEF WwW 78d oy J Plagues xt - Fraction —> Pecks. ‘ . ‘Suvyiving fa O97 Af O4F OFF (9-28 O10T O.1f 0.077 0.024 0.019 g.01F 0.005 . a —_— J atk 2 . OT Aysdys ti oF Fetes daactreins mm Got ~ Le’ (2 cpt UU Dote wo Seems No. of ° t& — 30a a bo 7s 9D g 180, +5 (Fo Trad. teed, -_ a 7 - » ) 1s iP ee ie if ie ‘s No. Sey” oO 6 / @ Z 4. | 6 % Se 4 as 33 6-0 o 6 6 6 oO aw PE naga Gre + me)” Y _— — _ — —_ —_ - lp _ c f — wm 1: eS! rn “9 Coven Aryees cf Warr Vg res attri Col f- Lr? _ 7 1 . 7 ab ‘¢ + 47 — 4p* ~— zi oO o 6 f oO — op 3 2 ( a ° > | tit. sq. 3st 348° | Procedure: Ul haw tt (weds ohrun i HET VO anda lubed lysate Ww Pearson Ditvivce = SO ew. — Brod ahi, a gem dos Ly 4B we vdlume | Cetet /0 wl Samp reanad at Ya ry | times. - HFT 7 she de = wy 3007 Assous D Claguas ou 8B 5 ~ WIS w2740 Dose 7 © 4 z 3 _ = _& 10° plogues Jk Gisot eS QL | e.7 . ST 03> “ond Trecha Tauvuiving : fe 0.7% os o-.53 ; 0.35 : 0.0/9 8. 007 a @Trousducls oB gol wa. fal cub heses oo: ee ., Recipemt Cobo Silt 0 Sto eh SF = _& -, Gey te wrsso (3) 107 ee SC (2) 107 gee BD SHE ORG SBD BRS Gar he, weir po? 3.3 SF bs - - .> — 2770 (0? BP 9. & - oo ~ —— . @) to’ =. Hb 3.5 Yr an ~ 3 2.7 Gets W234! wt BS ~~ ~ as-3 00 7 7 Ga, be Ww7s 1D” (Ob aD SD S.4 &-9 9.8 Ff Coke W2307 4-20 s t -1@ ~2 2 3 3 @ MM vclus Given howe been cowected tw S pry wens reverts of Mu wud’ cokw wtitnn. Ta fre Gilegy m WLI07 Figures Glue Gre papillac on lyse dddihir plot. — sprtauens rversucs g cpiilae . Nowe of frese peplles Word Che Ack fr Gok Grbese stololihy . (Gy Trea sdireny be! Gaye ref Cerys Teed | | | ! Nowe> FO oo 2 3 lot a & fe lpt 7 ° ° 4 ° oh, it 72¢ q| it Fe F ° ° Y Us ° L tt oo & £00 700 Bigs bff Joo 1¢ ae & ju ° ° were fs | Ova detes fo Gat byt Rectpens Wwrseo Miuaher beg bed Mo. Let No. 04. No. Net. Seq No. ¥. Rev, i Saucgle UV- Dore (wie) ° 2¢ ut if 6 9.& mi 24 uy (1 S { 8) Prada of fhe btusduchay pvedacced wih. lambda iwradtotd of sauaiibes, surwel 2 9.0X00~, (fF dHerewt Aausdutrar duakjieh abut 7 Sea req Guts bon Coed beled Ge le qoute and Gre artlele - Nu wile 7 bas ds Cuda adhe Eves eu vee Povey la dyace _fevown'y sey, gotten Let Lp" bet be® a ZF ? °o o o °o ° of G ° °o °° ° 2 @ Oo °o ° / ° f Ss g ° ° o o / G e ( oO Oo o . * / 3 / 0 o o / s” 0 ° 0 ° / 6 / ** ° oO Oo o t sf / ° ° / 6” ° ° 1 a thin Jeg. : 10 colonies refestea. Ail found Lp er ee awe be ome of These Sey reg anty geo slight lysis of be” tester gave slry Gt byes of bps fester, Shreaked out aud . ‘ A a bhree d th t-de sheds “A ut -4- wre chan os were ft’ fvaned Ow & re a ond Vom’ Ww yet she all tw. trausdudam plore, en Wor ols strenked on B are : obiente oh eames ee ey MME Fo oars pene ST aay Etpt 354 - 34h - 3r4B | fi fosm Prackyet of the taniduchrinn Pomel oir W'd (yee. be Mair tune Q number ot & ph oarerut vertu of Pee widicahws wee et auind Gu fouuk stable fv galore fermevttnin Oud unchtupet fr lambda peach. eget Lp guy pes gute dukvmined Ba fese Ae Otust bute Laub de t G berabha cnuthe HAAPe ewlhre , Thase kb AY rans dace os fo God Le” W2790 (Sr Sequqahuy us = Wt Sequegahug ) iA | UJ Dee oF IF 2 2 |. tf |_S & _ No. tush. ekamiied . —> zt 2% 23 2¢ ug 2¢ 4 Wo: fs PP Fuss. Fus | eas | ins dtus | ta dtu a bpt oo? o™ bur oo _ fee BS 8 ° y “3s ; Ls c ; ~ Le® > }12 5s IF SF Is ts Is a) —_ “ an las | ns Ions yr Sy oka neous reuevs ous \ G.6 7? A vag liqula > = Pe seul (iu Sau ple @)Tromduchia to Dad, lp W291 W Dok © | 2 3 Uf 7 6b No. CLormined ——-F Se oF ¢7 Na. Lev o Bas B8as. Let bs 2s ° 2us Lp 28 s 31s 6S tus t as Sag veg aby t7 se 10 / 2 0 0 « wt sesredaliy a + Go V3 2r zy Tg 7 7s : 44 73 Vt ¢ 7 o o Introduction fhe trans’er of genetic material between bacterial cells by temperate Salmonella and for Corynebacterium thee . In eah of these cases the transduction of genetic factors simely - bactephages has ‘een shown for certain has deldemonatratd. This mechansism of gentic recombination is in contrast with thcomlete saual mechanism of recombination in which the whole genetic materialof the cellparticipates at ane time. The study of these two mechhuisms and thed interrelatonship is difficult in biological systems in which only one has beenfywnd to oprate. The present report summarizes a study of EB. coli K-12 . where the ndependen oceurrence of sexual recombination ( Tatum and Lederberg, 1947) and fransductie recombination has been denoptrated.