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 =