Studies of novel transducing variants of lambda: Dispensability of genes N and Q

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glutination titer of CPV decreased gradually when kept at 4” more than 2 days. In order to confirm the above-menbioned results more precisely, the hemagglutination inhibition reaction was applied. Serial twofold dilutions of CPV antiserum were made in 0%ml amounts in 0.01 M phosphatebuffered (pH 6.8) saline, and a two-fold dilution without antiserum was set up as a control. The mixtures consisting of 0.2 ml of the virus suspension, 0.2 ml of the virus antiserum, and 0.4 ml of erythrocyte suspension were mixed and kept at 4” for 2 hours. The results obtained (Table 2) indicated that hemagglutination-inhibition tests are positive also. It appears that this is the first report of a successful hemagglutination reaction with insect virus. These studies will soon be published in their entirety. ACKNOWLEDGMENTS We are indebted to Prof. K. Hasegawa, Nagoya University, for his helpful advice, and to Mr. Y. Aichi-ken Agricultural Research Nakamura, Center, for his kind advice and technical suggestions. REFERENCES 1. HIRST, G. K., Science 94,22-23 (1941). 2. CUNNINGHAM, J. C., and TINSLEY, T. W., J. Gen. Microbial. 42, 397-401 (1966). d. HAYASHI, Y., and KAWASE, S., Virology 23,612614 (1964). S., Proc. Joint U. S.-Japan Sem.inar 4. K~WASE, Microbial Control Insect Pests l-6 (Shukosha Print. Co., Fukuoka) (1967). 5. KAWASE, S., and KAWAMORI, I., J. Invertebrate Pathol., 12, 395-404 (1968). 6’. MIURA, K., FUJII, I., SAKAKI, T., FUKE, M., and KAWAS~, S., J. Viral. 2,1211-1222 (1968). 7. MIYAJIMA, S., KIMURA, I., and KAWASE, S., J. Invertebrate Pathol. 13, 296302 (1969). SHIGETOSHI MIYAJIMA Basic Research Division, Aichi-ken Agricultural Research Center, Nagakute, Aichi-ken SHIGEMI

Faculty of Agriculture, Chikusa-ku, Nagoya, Accepted July

Nagoya Japan

25, 1969

KAWASE

Ziniversity,

Studies of Novel Transducing Variants of Lambda: Dispensability of Genes N and Q

The development of bacteriophage lambda can be divided into early and late phases. ‘Three early genes, N, 0, and P have been found to be essential in vegetat’ive growth (see Fig. 1). Mutants defective in N have a pleiotropic effect on other X functions, including DSA synthesis (1, 2). N mutants have recently been found to be deficient in X mRNA synthesis after induction or infection (2, 3). Mutants defective in gene Q, another gene controlling lambda development, lack late mRiYA and protein synthesis (,2-4). Since one or two copies of Q are insufficient for normal lysis and phage production (5), N may be required to provide many copies of gene Q by stimulating replication, in addition to turning on Q directly (6, 7). It might be assumed that mutations in genes with the complex functions of N and Q would be extremely lethal. However, it is observed that most early sus mutants of lambda, including those in N and Q are exceptionally leaky (8-11). This could be due to one or more events, such as frequent misreading of the nonsense mutation itself, or the possibility that the early function can be replaced, though inefficiently, by other genes of either the phage or the host. The question of the absolute necessity of any one gene may be answered better by examining strains deleted for that gene. Three transducing phages, a Xgal-bio, a Xbio and a Xgal (Fig. l), were used for the study of the functions and necessity of genes N and Q in the life cycle of X. The full details of the isolation and characterization of these phages will be discussed elsewhere, but a brief history of each of the above transducing phages is necessary for understanding their phage properties discussed below. The Xgal-bio was isolated from the preexisting Xgal29 cltl (a derivative of Xdg29 immQa4,Ref. 11). A lysate was made from the defective lysogen R594(Xga129 dtl) by tehperature induction and reconstitution of the

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defective Xgal “head” lysate in vitro, with a defective “tail” lysate from R594(XsusAll cltl) (12, 13). This reconstituted lysate has a high frequency of transduction (HFT) for galactose and a low frequency of transduction (LFT) for biotin. A Xgal-bio phage @gal29 biol01) was isolated from this lysate by using an attX deletion recipient (ld.), the deletion covering the entire bio region and part of the gal operon (strain SA206). The selection was, in effect, a double selection for gal+ and bio+. On our eosin-methylene blue galactose (EYIBgal) plates, gal+ transductants do not appear on the background of a gal-att)\-bio deletion strain, such as SA206, unless excess biotin is added to the plate, or the strain is transduced for both gal and bio characters simultaneously (15). This gal+ biof transductant of SA206 has several interesting properties. The transductant when streaked out segregates at a very high rate gal-bio- subcolonies, resembling the segregation rate of X&o’s broken within the cI gene (16). Whether this phage is integrated into the chromosome or exists as an extrachromosomal replicon has not been clearly established. Relatively stable lysogens can be made either by introducing a helper phage, or by selecting for very rare single defectives on galactose minimal medium in the presence of avidin. The defective lysogens, both segregating and stable, are found to be missing phage genes H-J as in the original Xgal29 and also genes N and rex (phenotypitally), which are presumably replaced by the bio region. The stable lysogen is immune to lambda superinfection at 30”. When induced by temperature, both the segregating and the stable lysogens yield lysates which have no transducing activity. However, after in vitro reconstitution with tail lysates, transducing activity for both gal and bio is coordinately found at a frequency of about 1O-3-1O-z per induced cell. It should be noted that the presence of a normal Xatt+ region in the host does not change the properties of this phage from those found on Xatt- SA206. One may conclude that this transducing phage can be excised from the chromosome

349

and matured into an infectious particle, albeit at low frequencies, in the complete absence of gene N. We next examined seven Xbio strains that have previously been characterized as defective and missing the gene N and parts of the immunity region (16). Five of these when induced from the defective lysogens have some transducing activity on a biorecipient, strain R879 carrying the bioA-24 mutation (17). One of these five, XbioSO-7, was studied more extensively. Single lysogens of XbioSO-7 segregate bio- subcolonies rapidly; double lysogens with wild-type lambda are relatively stable biof heterogenotes (16). The bio end point of XbioSO-7 is within the bioC region (17) ; its lambda end point is within the cI gene (16). We have found that a lysate, made by ultraviolet induction of the defective lysogen at a concentration of 2 X 10s cells per milliliter, contains infectious defective biotransducing particles (lOj/ml.) which behave like the parent, Xbio30-7. Additionally, the lysate contains particles which can form small, clear plaques (104/ml.) on sensitive strains. The properties of this plaque-forming isolate (designated as Xbio30-7pf) indicate that it is a derivative of the original XbioSO-7 particle. With X* helper, it transduces only those bio- mutants which the parent XbioSO-7 does. Its abilit,y to lysogenize in the presence of wild-type helper as well as its inability to plate on a lambda lysogen indicate that it is sensitive to lambda immunity. By complementation tests with known clear mutants of lambda (cl, ~11, and clll), it fails to complement cl and cIII mutants but can complement cII. In addition, the phage cannot form plaques on ret-A strains. This is a property of some Xbio’s which have deletions extending beyond the int-exo-p region but are N+ (18). All these properties are consistent with the phage having a deletion extending from gene int to within cI (Fig. 1). The defective lysogen, R879(XbioSO-7)) is missing the N markers sus7 and sus53 by marker rescue. We assume that XbioSO7pf is also missing the N markers, but it is hard to make a rigorous test for this prop-

(A)

A PROPHAGE

(E)

x

MAP

TRANSDUCING

A

Gal KTE

F

PHAGES &t

Chl D

Bio cIb ABFCD

( A gol 29

cIi OP QSR

bio 101)

A

F

ott Bio cU ABFC OP QSR J ( A bio 30-7 1

A

F

J

(111 int dnlmd34c!I N

( A lmm434

Gal Ci’KT

SR

gal M3)

FIG. 1. (A). Map of prophage X and bacterial markers transduced by X. Phage functions are indicated below the prophage map. Bacterial markers: gaZKTE, genes necessary for galactose utilization; chlD, chlorate resistance during anaerobiosis, and bioABCDF, genes required for biotin biosynthesis. (B). Vegetative maps of transducing phages of X. Xgal29 biolO1 and kimm434 galMS are reported in this paper. Xbio.!?O-7 was reported by G. Kayaj anian (16).

erty since the phage is nondefective. However, the following observations indicate that N is absent from bioSO-7pf: A Xvir SUN phage should not plate on a X lysogen because N function is absent. Likewise, a wild-type X phage will not form plaques, because of immunity. However, if these two phages are cross-streaked on a lysogenic background, we expect lysis at the intersection of the two streaks, due to vir N+ recombinants. To exclude any recombinants where N+ gene derives from the prophage, the lysogen used (SA216) carried a prophage deletion entering from the gaE side and removing phage genes int through rex including N. Instead of wild type X, we used either the nondefective (N+) transducing phage, Xbio7%3 (16), or h&030-7p.f.

Neither Xbio72-3 nor XbioSO-7pj forms plaques on SA216. When cross-streaked with Xvir SUN, Xbio7d3 gives confluent

lysis in the croys streak, but XDioJO-71)j does not. Qualitatively, this indicates that, Xbio30-7fjj is unable to supply the N gene. il\ closer examination of the Xbio30-7i?f cross-streak does reveal minute plaques uot typical of xvi,r lysis. These are presumably due to recombinants between the two phages which can grow in the absence of N and in the presence of immunity. XbioSO-7pf arises apparently by a “suppressor” mutation within the phage which allows more efficient phage production in the absence of N. The fact that the plaques are small is not surprising since nondefective &o’s make small plaques even in the presence of gene N. In order to insure the absence of any type of extraneous particle which could help the two N defective phages, discussed above, to replicate, serial dilutions of lysates prepared from SA206(XgaZ29 biol01) and W602(Xbio30-7) were assayed for transductants on appropriate strains in the presence of anti-Xserum. The number of transductants was always proportional to the dilution, and those transductants found in high dilutions, where the probability of co-infection was extremely small, retain the properties of their respective parents. The addition of X+ in the above assays consistently reduced the transduction frequency by a factor of ten. Whether XbioSO-7 can effect its own maturation and liberation is not completely certain. It is conceivable that every cell which liberates XbioSO-7 might also contain one or more XbioSO-7pf derivatives, and that the parent defective phage can never mature completely on its own. The same argument can be applied to Xgal29 biolO1, where the derivative corresponding to Xbio30-7p.f would be unable to form plaques. Preliminary studies with a XgaZ indicate that gene Q is also dispensable for phage maturation and plaque formation. The isolation of this phage is outlined in Fig. 2. By this method of selecting galactosetransducing phages one can theoretically delete genes to the left, of S and R with concomitant insertion of the bacterial gal region (20). The transduetant to be discussed, W33.50 @galA inzna43/t) was isolated directly by

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STEPS I.

II.

Aro G

GolKT “.’ ‘SRA

-Aro

Gal-

att F

J

F

J

att int SRA

m. IX ta)

Bio ABFCD

N

G t KT Aro

Gal-

lmm434 OPQSRA t

att F

J

Bio ABFCD

Chl att

4 Gall Gal*

I Gol+ P.

Aro G

Gal K-T-

att int ” .‘SRA

F

J

lmm434 Gal+ OP KTE N

Chl D

att

Bio ABFCD

FIG. 2. Isolation of the transductant W3350 (~imrn4.94 g&23). Step I. MS5061 (Ref. 21) has a deletion from the midpoint of galE through and including prophage gene Q. II. MS5661 was lysogenized with ximm@J. III. The resulting lysogen MS5061 (Ximm.434) was induced by ultraviolet irradiation to galMS). IV. This LFTmake an LFT-gal lysate. (The arrows indicate breaks that would yield ximm.& gal lysate was used to transduce W3350 (galK-8 ga1T-1). Two integration schemes are possible (A or B). Scheme A results in the direct formation of a gal+ transductant, while scheme B requires that a second double recombinational event occur between the two gal regions before one can get a gal+ transductant. V. Map of the gal+ transductant W3350 (Aim&@ gafM3).

transduction of W3350 with an LFT lysate of MS506l(~inzm~~~). Two possible integration schemes that will yield gal+ lysogens by this phage are diagrammed in Fig. 2, steps IV A and B. The gal+ lysogen thus made rescues all lambda markers tested except sus mutants Q21, &7s, and Qil7. Lysis is observed to occur in an exponentially growing culture of this lysogen between 4 and 5 hours after mitomycin C is added (5 pg/ml). Titration of the lysate reveals a transducing activity of ~108/ml on W3350. The Xgal found in this lysate retain the properties of the parental XgalM.3 imm&. In nddition to the transducing phage, there is a smaller class of plaqueforming particles (-lO”/ml). These form minute plaques on sensitive strains and lysogens of XimmX but do not plate on lysogens of Ximm.&. Attempts to purify this phage result in further selection of variants that fern, larger plaques. In this case, since we find such a high frequency of transducing particles in the lysate (~lO*/ml), it seems justified to con-

clude that XgalM3 inam@4, in the absence of gene Q, excises from the chromosome, matures, and lyses the host. The plaqueformers are apparently secondary mutants that occur with low frequency in the lysate. We conclude that the effects of genes N and Q in the normal development of X are quantitative rather than qualitative. In a wild-type background, both genes are necessary for the production of enough phage per cycle to give a plaque, but neither gene is essential for the formation of any infectious particles. In phages deleted for either N or Q, selection for lytic growth results in the accumulation of “suppressor” mutations of unknown nature that can compensate to some extent for the deletion. Of all known genes, N and Q are special in that their main function seems to be the positive control of other genes of the phage. The existence of limited phage development in their absence is consistent with such a role. Whether genes 0, P, S, and R might likewise be partially dispensable has not yet been examined.

ACKNOWLEDGMENTS

The Ultrastructure

We are grateful to S. Adhya, G. Kayajauiau, and J. Shapiro for strains; and to S. Adhya, A. Campbell, and G. Kayajanian for discussion and criticism of these experiments. This st.udy was supported by Research Grant, 8573 awarded to A. Campbell by the National Institutes of Health, Division of Allergy and Infectious Diseases. One of us (D. C.) holds aUSPHS predoctoral traineeship from the Department of Biology, University of Rochester, Rochest,er, New York. REFERENCES K., Virology 26,489-499 (1965). A., Isa~cs, L. N., ECHOLS, H., and SLY, W. S., J. Mol. Biol. 19, 174-186 (1966). A., BUTLER, B., and ECHOLS, H., 3. SKALKA, Proc. Natl. Acad. Sci. U.S. 58, 576-583 (1967). 4. DOVE, W. F., J. Mol. Biol. 19, 187-201 (1966). M., and THOMAS, R., 6. D.~MBLY, C., COUTURIER, J. Mol. Biol. 32,67-82 (1968). 6. THOMAS, R., J. Mol. Biol. 22.79-95 (1966). 7. DOVE, W. F., Ann. Rev. Genet. 2, 305-340 (1968). A. M., Unpublished observations. 8. CAMPBELL, 9. NAHA, P. M., J. Bacterial. 93,592-596 (1967). M., and ECHOLS, H., J. Mol. Biol. 10. WILLARD, 32, 37-46 (1968). A., Genetics 48,409-421 (1963). 11. CAMPBELL, J., Proc. Natl. Acad. Sci. U.S. 55, 12. WEIGLE, 1462-1466 (1966). A. M., Bacterial. Proc. 125, (1967). 1s. CAMPBELL, A., 14. ADHYA, S., CLEARY, P., and CAMPBELL, Proc. NatZ.Acad.Sci. U.S. 61,956-962 (1968). 15. COURT, D. L., Unpublished observations. G., Virology 36,31341 (1968). 16. KAYAJANIAN, A., KAYAJANIBN, 17. DE:L CAMPILLO-CAMPBELL, G., CBMPBF:LL, A., and ADHYA, S., J. Bacteriol. 94, 2065-2066 (1967). 18. MANI,EY, K. F., SIGNER, E. It., and R.\~DIxG, Virology 37, 177-188 (1969). 19. COURT, D. L., LORUX, G., and ADHYA, S., Unpublished observations. 20. CIMPBELL, A., ADHYA, S., and KILLI~N, K., Ciba Symp. on Bacterial Episomes 12-28 (1969). 21. SHAPIRO, J. A., and ADHY.Z, S., Genetics (in press). 1. BROOKS, 2. JOYNER,

DONALD COURT KOKI S.4TO

Departmenl of Biological Sciences Stanford University Stanford, California 94SO6 Accepted July

29, 1969

Newcastle

of Replicating

Disease Virus in the

Chick Embryo Chorioallantoic Membrane The need for ultrastructural data concerning the replication of Newcastle disease virus (NDV) in organs became evident to us during a pilot study of mixed viral infections. Although its appearance has been described in negative stains (l-5) and tissue culture studies of cell surface interaction and viral penetration (G-8), only two widely spaced reports have appeared concerning the morphology of XDV in the chick embryo chorioallantoic membrane (CAM) (9, IO), and none in its natural host, the chicken. Our preliminary st.udies produced morphologic data not described in either report. Eleven-day-old chick embryos were injected via the allantoic sac with a 10U2dilution of chorioallantoic fluid containing the virulent CGn strain of NDV which had an infectivity of 108.5 TCID50 per 0.1 ml. Neither the eggs nor the NDV pool was known to be free of avian leukosis virus. Infected and control embryos were incubated at 37” in a humidified incubator, turned regularly and sacrificed after 6, 18, 24, 32, 36, or 44 hours. Pieces of CAM were immediately fixed in 2% glutaraldehyde, postfixed in 1% osmium tetroxide, and embedded in araldite-Epon; sections were stained with uranyl acetate and lead citrate. Samples of NDV-infected chorioallantoic fluid from the pool and infected embryos were cleared by low speed centrifugation and then centrifuged at 100,000 Q for 2 hours. Portions of the supernatant fluid and pellet were prepared for negative staining with phosphotungstic acid (PTA). The pellet was fixed, embedded, and stained as noted above. All layers of the CAM contained developing viral forms. However, replication was most striking in the mesodermal layer of embryos incubated for 32 to 44 hours after allantoic infection. Virions were less numerous in embryos harvested at earlier intervals. The following data were derived from embryos with the most extensive virus growth. Newcastle disease virus developed by