VIROLOGY 9, 293-305 (1959)

Ordering of Genetic Sites in Bacteriophage x by the Use of Galactose-transducing Defective Phages’

ALLAN CAMPBELL”

Service de Physiologic microbienne, Institut Pasteur, Paris, Pra,nce

Bccepted July 9, 1969

In the transduction of galactose genes by bacteriophage X, each trans- ducing particle is characterized by the effective absence of an interstitial region of genetic material. The exact posit)ion of one of the end points of this region is different for particles arising from different primary events but appears never to change during subsequent multiplication. From this it is concluded that the transducing particle probably does not arise by crossing over within a region of full homology between phage and bacterial genomes.

It has recently been established that the transduction of the galactose genes of Escherichia coli K12 by bacteriophage X (Morse et al., 1956a, b) proceeds by the formation of a new genetic structure which is part phage and part bacterium in origin (Arber, 1958; Arber et al., 1957; Campbell, 1957; Campbell and Balbinder, 1959). Considered as a phage, it can he classified as defective; it ca,n lysogenize, and confer immunity upon, a suitable recipient, but cannot initiate a complete lytic cycle in single in- fection. This defective phage lacks an interstitial region of phage genetic material (dg region; Arber, 1958.) If it had been studied only in bacterial strains which were not marked at the galactose loci, it would probably have been classified as a phage with a long deletion (Benzer, 1957). How- ever, it actually contains some galactose genes of the host instead of the missing phage genes.

i Some of the experiments described here were performed while the author was at the Carnegie Institution of Washington Department of Genetics, Cold Spring Harbor, New York. The author is indebted to M. Andre Lwoff for his hospitality during the author’s stay in his laboratory at the Institut Pasteur, and to MM. Francois Jacob and Gerard Buttin for helpful advice and criticism during the preparation of the manuscript.

* Fellow of the National Foundation. Present address : Department of Biology, University of Rochester, Rochester, New York.

293

294 CSMPBELL

One obvious question is whether the exact extent of the dg region is identical for all transducing particles. We have already reported (Camp- bell, 1958) that the answer is negative. For this, we made use of a group of genetic markers which are so located that the left-hand end point of the dg region usually falls among them. In the present report we will extend these observations and also apply a useful corollary to the fact that different transducing particles are missing regions of difl’erent extent; namely, that the collection of transducing phages can be used in mapping new mutants falling within the missing region for some, but not all, of the transducing particles.

In order to understand the results, one must distinguish between low frequency transduction (LFT) and high frequency transduction (HYI’). The subject is still difficult to discuss in very simple terms because one always approaches it from two different points of departure. Experi- mentally, one must deal with LFT first. Each strain which yields an HFT lysate is descended, ultimately, from an LFT event. However, we have almost no direct evidence concerning the mechanism of LliT; our ideas about it are derived from what we know about HFT, for which we have now a good working model supported by experiments. It will therefore perhaps be clearer to invert the chronological order and de- scribe HFT first.

In high frequency transduction, one starts with a cell carrying two prophages. One is the standard, active X prophage; the second is the defective, transducing prophage. When a culture descended from such a cell is exposed to ultraviolet light, both genomes are induced to multiply and to mature. Of the two types of particles thus formed, only t’he de- fective phage contain the genes for galactose fermentation. The lysnte will transduce the galactose markers with high frequency because it contains these defective phage which, under suitable circumstances, can lysogenize the appropriate recipient and confer upon it, by their pres- ence, the ability to metabolize galactose. The transduct’ions occurring in such experiments are performed by particles which are genetically descendants of the transducing prophage in the original cell.

In low frequency transduction, one start’s with a cell carrying one prophage. It is a standard, active X prophage; no transducing phage is present’. A culture is grown and induced to lyse with ult,raviolet. The lysate thus prepared will t’rnnsduce the gslnctose markers wit’h low frequency. The result of each transduction is a colony of cells which have acquired one or more galactose markers from the donor. The majority of such colonies are composed of cells which carry one active

ORDERING OF GENETIC SITES IN BACTERIOPHAGE h 295

and one transducing prophage, and which will give rise on induction to high frequency transducing lysates.

The important point is that, in low frequency transduction, the trans- ducing prophages isolat’ed from separate colonies do not necessarily have a common origin. Since no transducing phage are present at the beginning, they must arise sometime during the course of the experi- ment. Inasmuch as the event by which they are produced occurs in parallel experiment’s, it must also be able to occur more than once within a single experiment. It is therefore essential to study the genetic properties of transducing prophages which originated from the same event in low frequency transduction and also those which originated from different events.

We will present first the data from phage crosses which establish the order of the genetic markers used in these studies, and then proceed to the results of the transduction experiments.

MATERIALS AND METHOIXS

The general methods for study of galactose transduction are those previously described (Campbell, 1957; Campbell and Balbinder, 1959). The isolation of host-dependent defective (hd) mutants of bacteriophage X is also described elsewhere (Campbell and Balbinder, 1958). A com- plete report on these mutants will be postponed until our information is more complete. For our purposes here, we need only say that they are mutants which form plaques on one strain of Escherichia coli K12 (CSOO) and do not on another strain (W3350), whereas wild-type X phage produces plaques on both strains.

The strain C600 (Appleyard, 1954) was kindly furnished to us by Dr. J. Weigle. Strain W3350 is a gall- gale- strain, also received from Weigle. A nonlysogenic gaZz- derivative of C600 (strain B 322) was isolated from a culture of C600 which had been infected with a lysate prepared by induction of a gale- lysogenic homogenote (Morse et al., 1956b) after exposure of that lysate to an ultraviolet dose sufficient to reduce the survival of active phage to about 10p3. Strain 1140 is an Hfr (high frequency of recombination) strain descended from the Hfr of Hayes (1953), kindly furnished to us by Dr. G. Bertani. It is gal+ and requires methionine.

Phage crosses have been performed with C600 as host. An aerated culture grown in broth to a density of about 1 X log cells per milliliter was centrifuged and resuspended at one-third the concentration in 0.01 M MgSO,. It was then aerated an additional hour at 37” before adding the phages. After 20 minutes of adsorption, antiphage serum was

296 CAMPBELL

added, and 10 minutes later the culture was diluted 1: 5 X lo3 in broth. The culture was aerated 90 additional minutes at 37”, and the phage yield was plated. When two hd mutants were crossed, the yield was plated both on C600 and on W3350. The distance was calculated as the ratio

2 X (plaques on W3350) X 100 (plaques on CAOO)

The corrections for finite input of Lennox et al. (1953) were applied to all data. When an hd mutant was crossed with a non-hd small-plaque mutant, such as m5, the yield was plated only on C600 and the wild- type plaques were distinguished by their larger size.

The hd mutants have a selective disadvantage in mixed infection with wild type on CGOO. This makes it impossible to equate frequencies of recombinants with frequencies of recombinationF and the calculated distances should therefore not, be taken t’oo literally.

We shall have some occasion to speak of experiments in which X phages of different immune specificities were tested against each other. In these cases, one phage was always XimmX and the other XimmJs4, where the former is reference type X and the latter is a phage (X-434 hybrid) isogenic with X except for a region controlling immune specificity (Kaiser and Jacob, 1957.)

EXPERIMEICTAL

The map distances calculated from the results of two-factor crosses between various hd mutants are shown in Table 1. Since a reliable ordering of these mutants cannot be made on the basis of such data alone, three-point crosses have been performed, as shown in Tables 2 and 3. These data establish the following order3:

hdll-(hdlo, hdl)-(hd,, hd~3)-hd9-hdl.?-hd2-hd6-m6h-h-hd7-c-hd8-hd3-hd~

Scoring of hd Mutants in the Prophage State

The nine mutants from hdl, to hde are near the left-hand end point of the dg region found by Arber (1958). It was therefore of interest to test

for the presence of these mutational sites in the transducing prophage. To do this, one takes advantage of the fact that neither the hd mutants nor the transducing phage can form plaques on W3350, but that wild- t’ype X can. One has therefore in principle a very sensitive tool for finding

3 The pairs (h&o, hdl) and (h&, h&) are not resolved by these three-point test,s. The free recombination with outside markers observed here is presumably due to high negative interference (Streisinger and Franklin, 1956).

ORDERING OF GENETIC SITES IN BACTERIOPHAGE ), 297

TABLE 1

MAP DISTANCES OBSERVELI IN TWO-POINT CROSSES BETWEEN hd MUTANTSO

hd,l hd,,, hd, hd4 hdl, hds hd12 hdn hds hd, hds hdr

hdl,, 64 hd, 54 2= hd4 52 53 53 h&s 6l 23 hds 9’ 41 h&2 72 93 82 21 hd? 181 12’ 93 71 81 51 42 h& 12’ 2’ hdr 151 11’ hds 171 11’ 3’ hd, 201 171 193 131 21’ 17l 16’ 113 16’ 4* 0.72 hd, 191 191 20’ 10’

a The numbers given are map distances calculat,ed as described in the text. Superscripts represent number of determinations.

TABLE 2

THREE-FACTOR CROSSES BETWEEN DIFFERENT hd MUTANTS”

hd,, h&o hd, hd4 hdo M hd:! hd, h& hds hd, hd,

c hdl

0.83 (174 1.4 (449)

1.8 1.8 2.2 2.6 2.9 8 9 1 1.6

c hdr c h.d,z

0.43

1.2 (65) 1.8 0.52 (355)

1.9 (629)

1 I

I -

c hdz c hd,

0.50 0.11 (339) 0.37 0.13 0.41 0.089 0.42 0.15 0.42 0.16 0.56 0.14

0.10 2.1 0.22 6 0.77 5 9.2

1.3 0.13

0

1 3

-

h h&a .50 (902)

.o (301)

.l

h hdl

0.59 (818) 1.0

2.1 (539)

a The numbers are given as the ratio ++ c/+++ or ff h/+++. The total number of hd+ recombinants counted is given in parentheses; where no such num- ber is indicated, more than 1000 such recombinants were counted. All crosses were performed on C600 with approximately equal multiplicities of the two parents.

whether any wild-type X can be formed by recombination between a given hd mutant and a given transducing phage.

In practice, we have employed two methods. For both, the trans- ducing prophage to be tested must be the prophage of a defective lyso- genie strain of the type W3350(XgaZ). One can then either (1) spot this

298 CAMPBELL

TABLE 3

THREE-FACTOR CROSSES OF SINULE hd MUTANTS"

hd mutant

h& ++ hd, +c hd ++ hdz +c h& +f ++ hd, fc hd,

0.52 (155) 0.65 (28) 2.1 (68)

0.60 (40) 2.2 (55) 3.3 (39)

0.36 (133) 1.9 (112) 13 (14)

0.12 (106)

a The numbers are the ratios ++c/+++ 01’ ++co?/+++. The total number of h/J+ txj+ 01’ hdf Vllj+ plaques counted is given in parentheses. All crosses were performed on C600 with nppu)simately equal mult,iplicit.ies of the two parents.

strain on a plate seeded with a mixture of an hd mutant and strain W3350 and irradiate t,he plates with IJV in order to destroy super- infection immunity or (2) cross-streak t’he strain ag&st Xhd of a different immune specificity. In the first case, one looks for lysis of the back- ground W3350, and in t,he second case, for that of the strain W3350 @gal). This is equivalent to looking, in both cases, for the formation of hd+ recombinants, which in the second case must also be of t,he proper immune specificity to grow on the defective lysogen used.

A control experiment is to use, instead of W3350(XgaZ), strains of the type W3350(hhd) of the appropriate immune specificity. The result’ of such experiments is that’ a mutant hd, gives a positive result with W3350(Xhd,) if and only if m and n are different, for all pairs of the thirteen mutants used.4 This control is quite adequate for our purposes here; and the question of whether we are really testing by t,hese methods for a mutational site or for some larger unit which contains it, such as a cistron, need not bother us for the moment.

Galactose Transduction

Most of our data have been obtained by t’he second method. The hd mutants have all been isolated from kimmA, and from only three (hdl, h&, hd:]) have we isolated from further crosses the phage Xhd imm434. Therefore, we used for most of the low frequency transductions a phage

4 All pairs have been studied by the first method, and only some pairs by the second.

ORDERING OF GENETIC SITES IN K4CTERIOPHAGE x 299

of the type Xhdf irnrnJz4, so that we could later perform tests with all of the hd mutants. We isolated and purified the heterogenotes produced from these transductions; these generally carried both an active prophage hhdf imm434 and a defective prophage Agal+ imm434. The presence of the former makes these strains able to support the growth of all of the hd mutants, regardless of the genotype of the host or of the transducing prophage; so we next induced these primary lysogenic heterogenotes to prepare high frequency transducing lyxates, and then used these for transductions with W3350 as recipient. These transductions result in three principal types of colonies: (1) lysogenic heterogenotes, W3350 gal- (Xhdf imm434) (Xgal+ imm4”4); (2) defective heterogenotes, W3350 gal- (Xgab+ imm434); and (3) phage-sensitive haploids, W3350 gal+.

We then tested with the hd mutants a number of such independently isolated strains produced by transduction. As expected, all t’he mutants gave positive tests with all the lysogenic heterogenotes, and negative with all the sensitive haploids. It is only the defective heterogenotes which tell us the genetic content of the transducing prophuge.

The results, shown in Table 4, allow two conclusions: (1) Among the transductions which were obtained using any one lysate, the defec- tive lysogens are all identical in their content of hd sites. (2) The trans- ductions obtained using different lysates, which originated from sepa- rately isolated primary lysogenic heterogenotes produced in low frequency transduction, are often different.

Ordering sf Genetic Sites bg Use of the Transducing Phages

A third, and very comforting, conclusion can also be drawn from the data of Table 4. This is that the group of sites missing from any one transducing phage is coherent. This fact is of course implied in our manner of recording the data in the last column of this table; in any other case, we would be compelled to itemize the missing sites indi- vidually. One can actually turn the argument around and deduce the order of the hd sites by assuming such coherence for all the transducing phages of Tables 4 and 5. One thus obtains a unique order which is compatible with the transduction data:

hd,-hdlo-hdl-(hd4, hdla)-hd,-hdr(hds, hds)

The validity of the assumption follows from the agreement between this order and that determined from phage crosses. This manner of ordering is the same as the “overlapping deletions” method of Benzer (1957).

300 CAMPBELL

TABLE 4

TYFES OF TRAKSDUCIXG PHAGES OBTAINED~

Strain induced to prepare LFT lysatc

CSOO(X imm”~“) d 226

7% 174

Primary recipient

#322

d 322

w3350

Number of Number of

primary defective hd+ Alleles abseI~t~~ hetero- lysogens genotes tested

49 43

4 11 16 30 25

21

hdy-hda hdr-hde hd-hdcj hdg-hds hds-hd, hd4-hds hd,-hd, hd,urhds hd,,-hd6

C600(hhdlittlnc43') A322 1 1

I322

w3350

# 322

2 1

1

35

20 hdl, hdg-hds 14 hdlo-hd, 13 hd,,-hd,

16 hdl, hd,

13 hd, 10 hd,, hd,

3 hdz, hd:,

302

D Experiments as described in the text. The two different LFT lysates from C60O(X&~rr~~“) were from two independently lysogenized derivatives of C600. The hd mutants will grow on strains CBOO and 322, and not on Ii40 or W3350.

h The defective lysogens derived from Xi~~7a~~~ were tested against all 13 hd mutants, and those from hinlm’ against hdl, hdn, and hd,. For those from Xinm434, the region missing is indicated. For example, on the defective heterogenotes of the first row, the alleles hdll+, hdlo+, hdl’, hdJ+, and hd,,+, as well as hdT+, hda+, hd,+, and hdjf were present, whereas hdy+, hdll+, hdz+, and hdG+ were absent.

Transductions by hd Mutants

One assumption that is basic to the above argument is that when a transducing phage appears, by the tests used, to contain an allele hd,, that this allele is genetically descended from that of the phage used in making the LPT lysate. One might also imagine other origins. E’or example, a piece of the bacterial chromosome might, for all we know,

ORDERISG OF GENETIC SITES IN BACTERIOPHAGE 1 301

perform the same function; and if this piece of bacterial chromosome happened to be located close to the galactose loci and to be sometimes included within the transducing phage, we might be quite seriously misled.

The best evidence that we are studying the extent of the phage genome present is that, whenever the phage used in preparing the LFT lysate has been a mutant hd,,, the defective lysogens ultimately ob- tained always behave as though they did not have the hd,+ allele. Some of the data are given in Table 4. Especially significant is the one transduction performed with Xhdl irnrnJs4 which gave rise to defective heterogenotes containing the alleles hdd+ and hdla+, but not hdl+. No transduction with Xhdl+ imm 434 has ever given this result, which would imply the absence of a noncoherent group of sites.

One technical point should be mentioned in connection with this ex- periment. We have stated earlier that the transductions resulting from the use of an HFT lysate may be truly lysogenic, defective lysogenic, or phage-sensitive. We retain this purely operational classification even now that we are fairly certain that the basic distinction is be- tween double lysogens, which carry a transducing prophage plus a second prophage; single lysogens, which carry only the t,ransducing prophage; and completely nonlysogenic strains.

The distinction clearly becomes more important in these experi- ments, because if the phage is an hd mutant and the recipient is W3350, both the types W3350(Xhd,)(XgaZ+) and W3350(XgaZ+) are defective lysogens. They are however distinguishable by the fact that the former prove to contain, by our tests, the hd+ alleles for all the mutants except h,d,. The fact that this expected result is realized indicates, as far as it goes, that the hd markers behave as true markers and do not interfere with the mechanics of the transduction process in any way which might make our other conclusions spurious.

Galactose-negative Segregants

The transducing prophage does not form completely stable lysogens. About once in every lo3 cell divisions, it is lost from the cell. The result of this loss is generally the formation of W3350 gal- @gal+) or of W3350 gal- (X) from W3350 gal- (X) (XgaZ+). Most of the galactose-negative segregants isolated from such cultures prove to be of this type. Less common are the homogenotes W3350 gal- (Agal-) or W3350 gal- (A) (Xgal-).

We have examined 181 gal- segregants from strains of the types

302 CAMPBELL

W3350 gal- @gal+ imm4a4) for the hd+ alleles. Of these, 165 were phage- sensitive and contained no hd+ alleles by our tests, whereas 16 were immune to Ximm4”4 and contained hd+ alleles at all the same sites as their parent strain. This shows that any imaginable process by which these hd,L+ alleles might be occasionally “left behind” in the bacterial cell cannot occur at a frequency detectable by this test.

Cal- segregants from defective lysogens of, the presumed type W3350 gal- (Xhd, immSd4) @gal+ irnrn”““) have also been studied. In this case, the gal- segregants contain hd+ alleles at all sites except h&. This is the expected result for both the predicted t,ypes of segregants W3350 gal- (Xhd,, imm434) and W3350 gal- (Xhd,, imm434) @gal- imm434).

Relation between Physical and Genetic Properties of the Transducing Phage

The results report.ed here demonstrate that one genetic property of the transducing phage, namely, the l&hand end point of the dg region, is determined somet’ime before the isolation of the lysogenic heterogen- otes produced in low frequency transduction and rarely if ever changes subsequently. Weigle et al. (1959) have shown t’hat another property, namely, the density of the transducing phage as determined by centrifu- gution in a cesium chloride gradient, behaves in exactly the same manner. It, is of imerest, to know whether the two properties are related, and one obvious experiment is to measure both properties for the same set of t~ransducing phnges.

TABLE 5

TESTS OF INI~EPENI)EST HETER~CEN~TES FOR WHICH DENSITY -MEASUREMENTS ARM AVAII.ABLE

Lysoyenic heteroyenote ICumber of defective hcteroyenotes tested

hd+ Alleles absent

1 23 hdh-hda 2 18 hdh-hdse 3 2 hds-hd, 4 23 hd4-hds 5 1 hdn-hds 6 7 hdrhds 7 6 hds-hds 8 20 hdrhds

10 30 hd4-hds

G The het,erogenotes from #2 gsve wenk spots with hdl and hdlo, which indi- cates thnt the end point in igi2 is further to the left thttn that of # 1, f4, and #: 10.

ORDERING OF GENETIC SITES IN BACTERIOPHAGE k 303

Dr. J. J. Weigle has kindly furnished us with ten lysogenic heter- ogenotes for which the density measurements had already been made. One of these ten strains ( # 9) was unfortunately lost during handling. The results from the ot,her nine are shown in Table 5.

According to our data, one can thus place the nine heterogenotes in the order:

5-(3, 6, 7)-(1, 4, 10)-2-S

where in I5 the end point is furthest to the right, and in B 8 it is furthest to the left. If the differences in density between transducing particles observed by Weigle et al. were attributable solely to the amount of phage genetic material missing on this end, one would expect, that heterogenote R 5 would yield the most dense transducing particles and d 8 the least dense. Actually, their data place the heterogenotes in the order (from most dense to least dense):

5-Z-6-1-10-7-8-4-2

The apparent correlation is probably meaningful, but the data are not extensive enough to prove the point. What is clear is that there is not a complete correlation. Inasmuch as the accuracy of both the physical and the biological measurement,s virtually precludes any error in either order, one is forced to one of two conclusions. Either (1) the density is a direct function of the position of the left-hand end point measured by genetic means, but the function is fairly complex and not monotonic, or (2) there is more than one independent variable in the process by which the transducing particle is formed.

Logically, it is impossible to exclude absolutely the first alternative with any finite number of measurements.5 We consider the second as much more likely from the data presented here. The second variable is probably either the amount of bacterial genetic material transduced or the position of the right-hand end point of the dg region on the phage genetic map. Since Arber (1958) has concluded that the right-hand end point is rather sharply determined, it is probable that the amount, of transduced bacterial genetic material can vary. This conclusion will

6 This is the logic of functions whose domain is a continuous series of points. That the points on a genetic map are not continuous at the level of fine structure now experimentally obtainable is indicated by the work of Benzer (1957). There is no obvious reason why the hd sJTst,ern cannot eventuallp be perfected to a simi- lar level of precision. If one could then obtain two transducing particles with different densities and precisely the same end point, this result would rigorously exclude the first alternative.

304 CAMPBELL

probably hold even if the observed density differences turn out not to be ascribable directly to the amount or density of DNA present.

1>ISCUSSION

The principal conclusion to be drawn from the present investigation is that transducing particles derived from separate LFT events are genetically different, whereas those derived from the same event tend t,o be the same. The latter fact is predictable from what we already know about HFT. It provides reassurance that all the transducing particles from a lysogenic heterogenote are, in fact, genetic descendants of the same transducing prophage.

The actual result goes, however, somewhat further. Among the 4:32 strains examined in Tables 4 and 5, the rule that transducing phages coming from a common ancestor contain t’he same amount of phage genetic material is obeyed without exception. This suggests that there is no mechanism by which this property can change at all frequently. If this is true, it provides rather strong limitations on the number of possible modes of origin of the transducing particle; and in particular it rules out any models which assume simple crossing over between phage and host chromosomes at various points within a region of true homology between the two. We shall discuss further evidence on this question elsewhere.

The use of overlapping deletions has been very valuable in fine- structure mapping. When, as in the present study, the end points of the deleted regions of a collection of transducing phages lie within the region of interest, these phages provide a very convenient source of such de- letions. Thus far, the delct.ions of all the galactose-transducing phages examined have a common core, which includes the sites hdz and h& and also the h locus.

It will be of interest t’o know if a similar situation obtains in other transductions which are mediated by defective phage particles, such as the transduction of lactose by phage I’1 (Luria et al., 1958). It must be pointed out, however, that the present work was possible only be- cause of the extreme facility with which one can test for the presence of an hdf allele. To perform the crosses described here without the use of a series of markers which can be selected for in this manner would be an ext’remely laborious task. It was simply good fortune that we had such markers for the X-gal system, and it would be unduly optimistic to expect all other systems to be as fortunate,

ORDERING OF GENETIC SITES IN BACTERIOPHAGE 1 305

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