CHROMOSOMAL REARRANGEMENTS IN ESCHERZCHZA COLZ STRAINS CARRYING THE CRYPTIC LAMBDA PROPHAGE'

V. ZAVADA2 AND E. CALEF

International Laboratory of Genetics and Biophysics, Naples, Italy

Received May 24, 1968

T H E insertion of h into the chromosome of Escherichia coli K12 increases the distance between the loci gal and bio, bracketing the prophage. Thus the

frequency of PI mediated joint transduction of gal and bio from a lysogenic donor is considerably lower than that from a non-lysogenic one (ROTHMAN 1965). Accordingly, the frequency of gal-bio cotransduction from a donor carrying a deletion in its prophage should be intermediate between the frequencies found with lysogenic and non-lysogenic strains.

prophage the region between genes N and R, including the immunity region, is deleted (FISCHER-FANTUZZI and CALEF 1964; CALEF and FISCHER-FANTUZZI 1965; and MARCHELLI, PICA and SOLLER 1968). Contrary to what should be expected in the case of a mere deletion in the prophage, no P1 mediated gal-bio cotransduction has been observed from the cryptic lysogen U1 60. In addition, no viral genes were transduced with gal and only those situated near bio were transduced with it (ROTHMAN 1965). Thus the deletion in the cryptic prophage present in U160 seems to result from a more complex reorganization of the host chromosome. It was suggested that a chromosome segment was translo- cated between gal and bio, spacing them enough to preclude joint transduction (FISCHER-FANTUZZI, 1967; and ZAVADA and CALEF 1967).

The aim of the present work was: 1) to find whether the absence of P1 medi- ated joint transduction of gal and bio observed with U160 is a general property of strains carrying a cryptic prophage between these loci, and 2) to detect possible chromosomal rearrangements in cryptic lysogens negative in gal-bio cotransduction. Thus several independent cryptic derivatives were analyzed for gal-bio cotransduction and kinetics of chromosomal transfer. The latter phenome- non was investigated by means of interrupted mating experiments with cryptic Hfr males unable to donate simultaneously gal and bio in PI transduction.

In the cryptic

MATERIALS A N D METHODS

Bacterial and bacteriophage stocks: Cryptic lysogens used in this work were independent isolates derived from several ancestor strains lysogenic for crg, a X phage originally present in U146 and containing a cryptogenic factor (FISCHER-FANTUZZI and CALEP 1964.; CALEF and FISCHER-FANTUZZI 1965; and MARCHELLI et al. 1968). In Q+R+ cryptic lysogens, the deletion in the prophage did not extend over the genes Q and R, in the rest of cryptic strains, both genes

Work carried out under the association Euratom-C.N.R.X.N.E.N. Contract No. 012-61-12 BIAI. ' Present address: Dept. of Microbiology, Faculty of Science, Charles University, Vinicna 5, Prague, Czechoslovakia.

Genetics 61 : 9-22 January 1969

10 V. ZAVADA A N D E . CALEF

TABLE 1

List of bacteria lysogenic for X cry

Bacteria Type of X cry Name

U3 7 cry Q+R+ 1 U37 cry A, B, c 708 cry Q+R+ I , % 708 cry A, B, D, E, F, H, K C600* * cry Q+R+ z1, z2,23,223 C600* * cry Z7,Z11,Z12,Z15,221,Z26,227,Z28

Z4*, B*,ZlO*, Z17*, Z18*, Z19*, Z O * , Z25*

* Linked gal-bio donors by PI transduction ** The cryptic derivatives of C600 were obtained from Dr. L. PICA and Dr. C. MARCHELLI to

whom thanks are due.

were missing. All cryptic strains were gal+ bio+ and were derived from the following ancestors: U37 = Hfr H, 708 = AB311 (TAYLOR and ADELBERG 1960), C600 (APPLEYARD 1954). The list of the cryptics is given in Table 1.

U335 is identical with W602 of ROTHMAN (1965), F- B,- leu- gal- bio- str". U499 was ob- tained by lysogenization of U335 by X CIII sus54 sus9 sus24. U2M is F- gal- trp- str". U279 (CALEF et al. 1965) is pm-, i.e. non-permissive for sus mutants of A. C600 and its streptomycin- resistant derivatives U154 are pm+.

The phage strains h susB1, X sud6, h susG9, X susKZ4 and X susR54 were used in marker rescue tests. The strain X CI 60 was used to check for immunity. Plkc (LENNOX 1955) grown on U335 was used for infecting the gal+ bio+ donors.

Media: The concentrations are given in grams per liter. Tryptone broth and tryptone agar: tryptone Oxoid (IO), NaCl ( 5 ) , thiamine-HC1 (0.001), agar (15), p H = 7. LC agar (LURIA, ADAMS and TING 1960). Bacto-Penassay medium Difco. Minimal agar medium was the minimal medium of DAVIS and MINGIOLI (1950) supplemented with: thiamine-HCl (0.0001), K,HPO, (2), Bacto-eosine Y Difco (0.04), Bacto-methylene blue Difco (0.065), streptomycin-sulphate (0.2) and Oxoid-agar (15).

The gaZ+ transductants and recombinants were assayed on minimal agar medium supple- mented with Bacto vitamin-free casamino acids (2.5), biotin (0.0001) and galactose (IO). When used to detect the gal+ progeny in crosses with U264, the medium was supplemented also with L-tryptophan (0.01). For direct assay of gal+ bio+ cotransductants biotin was omitted. The bio+ transductants and recombinants were assayed on minimal agar medium supplemented with Bacto vitamin-free casamino acids Difco (2.5) and glucose (2.5). The minimal agar medium supple- mented with casamino acids (2.5) and glucose (2.5) was used for assay of trp+ recombinants. The leu+ recombinants were assayed on minimal agar medium supplemented with Bacto leucine- assay medium Difco (l), glucose (1) and tmethionine (0.005). SM medium (WEIGLE et al. 1959) was used to dilute bacteria and phages. EMB-galactose agar was prepared according to LEDFXBERG (1950).

crg grown overnight in tryptone broth plus anti-X serum from a small inoculum (ca. 103 cells per tube) was plated on EMB plates containing 0.05% glucose plus anti-h serum and irradiated with a UV dose giving several hundreds of survivors per plate. The plates were incubated overnight and clones of cured cells were detected as dark red sectors in pale pink colonies (FISCHER- FANTUZZI and CALW 1964). They were reisolated on tryptone plates containing anti-X serum and the non-immune isolates were checked by the marker rescue test to detect the cryptic lyso- gens. The cryptic strains derived from separate tryptone-broth cultures of the ancestor lysogen are considered to have arisen independently.

Procedure for P I transduction was described previously (ZAVADA and C a w 1968). The

Methods: Isolation of cryptic derivatives: 0.1 ml of the ancestor culture lysogenic for

THE CHROMOSOME OF E. coli (Xcry) 11

direct assay of gal+ bio+ cotransductants was used to measure the frequency of joh t transduction of both markers. The ratio between the frequency of gal+ bio+ cotransductants and that of either gal+ or biu+ single transductants has been defined as the relative frequency of CO-

transduction. Interrupted mating experiments and analysis of the progeny: Parent cultures were grown in

Bacto-Penassay medium from a 10' cell/ml inoculum for 4-5 hrs at 37°C. Only the recipient culture was grown with aeration. 30 min before mixing with the recipient the donor was diluted two-fold in prewarmed Penassay medium. Equal volumes of both parent cultures were mixed at 0 min. After this manipulation the concentration of the donor in the mixture was a b u t 1.108 cells/ml and the ma1e:female ratio about 1:5. Two minutes later the mixture was gently diluted ten-fold in prewarmed Penassay medium. 0.2 ml samples were withdrawn at intervals, diluted ten-fold in ice-chilled SM medium and shaken vigorously to interrupt the conjugation. Several dilutions were plated on the respective media and the progeny carrying the selected alleles of the donor were scored after 30-40 hrs at 37°C. The transductants and recombinants were purified by restreaking on the respective selective media, transferred on tryptone plates and replicated to score for the non-selected alleles of the donors and the allelic state of the prophage present.

EXPERIMENTAL

The PI mediated joint transduction of gal and bio from donors carrying the cryptic prophage: The relative frequency of gal-bio cotransduction was esti- mated with 34 independent cryptic isolates listed in Table 1 . The recipient was U499. With 26 of them, the relative frequency of gal-bio cotransduction was less than 0.1 %, if at all. The frequencies of either gal+ or bio+ single transductants were about the same and of the order 10-6-10-7. On the other hand, with cryptic strains Z4,28, Z10, Z17,218,Z19,Z20 and 225, the relative frequency of gal-bio cotransduction was approximately 10%. The analysis of transductants was per- formed with Z19, 220 and 2 2 5 . and the results given in Table 2 show that the cryptic prophage is situated between the gal and bio loci of these donors.

The absence of PI mediated joint transduction of gal and bio thus seems to be a frequent but not an obligatory character of strains harboring a cryptic prophage between those two loci.

The kinetics of chromosomal transfer from cryptic Hfr H males and thegenetic constitution of the progeny: The Hfr H derivatives U37 (X crg), U37cryQ+R+ 1, U37 cryA, U37cryB and U37cryC were studied by means of interrupted mating experiments. The females used were U499, U335 and U264.

The kinetics of chromosomal transfer from U37 ( A crg) was that typical for an Hfr H male. As illustrated on Figure 1 , leu entered the zygote a t about 9 min and gal and bio ca 18-19 min later. As shown in Table 3, close linkage between gal and bio could be observed among the progeny selected for the distal marker.

The cryptic males U37cryQ+R+ 1 and U37cryC transferred their chromo- somes with similar kinetics. These kinetics were partly different from that found with U37 ( A crg) . While the entry times for leu and bio were unchanged, gal entered the zygote much earlier, preceding bio by 6 min (Figures 2 and 3). As follows from Table 3, the linkage between gal and bio was still high among the bio+ progenies. The majority of gal+ bio+ recombinants were cryptic lysogens, i.e. the cryptic prophage is obviously situated between the loci gal and bio in the respective males. Unlike U37 ( A crg) and its cryptic derivatives A and B, the cryptic males Q+R+ and C were lac-.

12 V. ZAVADA AND E. CALEF

TABLE 2

PI mediated transduction of gal and bio from cryptic donors io U499. The genetic constitution of transductants.

~~ ~ ___________

Transductants Relatwe frequency of gal-bio

Donor Selected marker gal imi 91: 24t bio Number cotransduction

z-19 gal + bio + - + 37 + . + 1(d)

2-20

38

+ + 4 10% gal + -

+ 3 33

- - - - -

40

bio + + - + 9 22% + 16

3

- - + 12 -

+ + - -

+ + +

gal -t bio + -

gal+ - + + - -

- -

- - _

bio+ + - + + - + + - + - - -

’ ( + + . ) + Z-25* gal+ bio+ -

40

40

4 10% 2 2

32 40 -

3 7% 17 6

14

40 -

38 2

40 -

5 12% 4 5

26 40

bio+ + - + 5 12% - - + 21 + 5

9 40

- + + - - -

(d) = defective * This strain bears the stock culture name of U476A. +The symbol im stands for lysogenic immunity. The presence of immunity was routinely

$ 9 and 24 are referring to the mutants susG9 and “24. scored by cross streaking the bacterium to be tested against a streak of X CI.

THE CHROMOSOME OF E. coli (Xcry) 13

1(

1

0.1

0.01

FIGURE 1.-Kinetics of chromosomal transfer from U37 ( A csg) to U499.

With the cryptic males U37cryA and B two different transfer kinetics were observed.

As follows from Figures 4 and 5 , the cryptic male B transferred its chromosome with an Hfr H orientation, the sequence of entries being: leu-gul-trp, with leu entering at 9 min. Unlike U37 ( A crg) both gal and bio were transferred much earlier and the frequencies of both respective progenies were greatly reduced, the reduction being higher with the gal+ progeny. The entry time of the distally situated trp marker was again that reported for Hfr H (TAYLOR and TROTTER 1967) and the frequency of the trp+ progeny was not reduced. All the gulf progeny from the cross with U499 was bio- and vice versa and the alleles of the cryptic prophage could be rescued only from the bio+ progeny (Tables 3 and 4). A similar situation was found in the cross with the non-lysogenic female U335: the frequencies of gal+ and bio+ progenies were reduced similarly as in the cross with U499 and all 80 guZ+ recombinants tested were bio- and did not rescue susl 1 +. Among 80 bio+ recombinants only 3 were gal+ and rescued susl 1 +, 15 were gul- susl 1 + and the rest was gal- and did not contain sus1 1 +.

The male U37cryA even after repeated cloning transferred gal as early as leu and at a high frequency. The early gal+ progeny sampled after 10-15 min of

14 V. ZAVADA AND E. CALEF

U)

c m K

c.

.- n E 0 U aJ L

.c 0

s

1

1

0.1

0.01

U37 cry Q'R'XULSS

10 15 20 25 30 35 40 m FIGURE 2.-Kinetics of chromosomal transfer from U37cryQ+R+ to U4.99.

mating was sensitive to the male specific RNA phage f2 (LOEB 1960) but unlike U499 carrying the Fl-gal+ of SIGNER, BECKWITH and BRENNER 1965, our strains retained their gal+ character after overnight growth in try-ptone broth con- taining up to 10 p,g/ml acridine orange. As shown in Table 4, the leu and bio alleles of the male and the alleles of its cryptic prophage were absent from the gal+ progeny even after prolonged period of mating. The transfer of ku and bio occurred with a time sequence typical for Hfr H but the frequencies of the respective progenies were reduced significantly (Figure 6). As follows from data given in Table 3, the linkage group gal-cry-bio still exists in U37cryA. The majority of the gal+ bio- progeny from the cross U37 (Acrg) x U499 were defective lysogens. Remarkable amounts of defective lysogens were observed under similar conditions with cryptic derivatives of U37 too. The defect in the prophage of such gal+ bio- recombinants could not be identified using the set of h sus mutants covering the genes A through R. Consequently, by the term defec- tive prophage we only mean a prophage which does not produce any plaque- €o&g particles upon UV induction. Here, it should be noticed that the appear- ance of these defectives depends on the selection of crossovers with exchange on the gal side of the prophage; indeed, the progeny of the same cross, where ex-

15 THE CHROMOSOME OF E. coli (Acry)

1c

1

0.1

U37 cry C xu499

i

FIGURE 3.-Kinetics of chromosomal transfer from U37cryC to U499.

change was selected on the bio side (biof progeny), contained much fewer defectives.

Note added in proof: The defectivity of the h sus prophages was due to the absence of an active suppressor in the respective gaWrecombinants which in- herited the pm- alleles of the non-permissive male.

DISCUSSION

The lack of the P1 mediated joint transduction of the loci gal and bio from a donor lysogenic for a cryptic h prophage is clearly a frequent though not a general phenomenon. Out of 34 independent cryptic lysogens investigated, 26 were nega- tive in gal-bio cotransduction, similar to the strain U160 studied by ROTHMAN (1965). On the other hand, cotransduction of gal and bio occurred with 8 cryptic donors from which at least three harbored the cryptic prophage between the respective loci. Among independent cryptic isolates derived from a common lysogenic ancestor cotransduction-positive and cotransduction-negative donors were found, as illustrated by cryptic derivatives of C600 ( h crg) .

To explain the lack of gal-bio cotransduction from a cryptic lysogen, it has been suggested that the missing viral genes might have been replaced by a longer segment of chromosomal material (FISCHER-FANTUZZI 1967; and ZAVADA and

16 V. ZAVADA A N D E. CALEF

TABLE 3

Genetic constitution of the bio+ progeny of crosses of U499 with U37 (A crg) and its cryptic hriuatiuesf

Donor leu gal im 54' 9' 24' 6' Number

U37 ( A erg)

U37cryQ-R- A

+ + + + + + - + + + + + + - + + + + + + + + _ _

+ - + + "

_ _

+ + - ' . + + - + - + + + + - . . + -

. . + + - - - + + + + - + + + + - + +

- -

- _ _

- - -

U37cryQ-R- B + - + + + + + + + + + + - + + - + - + + + + - + + + - +

_ _ - -

- _ -

- - _ - -

U37cryQ-R- c + + - + + - + - + + + + - + - + - - f - - + + + - + + + + + + +

+ + - + + +

_ - -

t - _ _ _

- _ -

- - _

- - - -

74 39 2 3 1 1

120 -

49 14 1 1 8 1 1 1 8 1 (d)

85

1 1 31

1 6 3

10 13 44.

120

1 (d)

-

70 28 4 1 1

THE CHROMOSOME OF E. coli (hcry) 17

Donor leu

U37cryQ+R+ 1 + + -

- - + -

gal im 54' Y*

+ - + + + - + + + + + + + + - + + + - + - + - + - +

- _

24' 6' Number

+ + + + + + +

70 39

3 1 1 1 2 + - + - + - 1

- - + - + - I - - + - - + 1

1 20 -

(d) = defective * 54,9,24 and 6 are the numbers of the sus mutants used in the cross. t I n t e k p t e d after 60 min of mating.

CALEF 1967). Exactly this is true with the two cryptic derivatives of Hfr H: U37cryQfRf 1 and U37cryC. As follows from the kinetics of chromosomal trans- fer, approximately $$ of the chromosomal material situated normally between 2eu and gal has been transposed between gal and bio in those cryptic males. The

1[

D z 1 m

E

c 13

0 U W L

.c 0

.^

0.1 s

0.01 5

U37 cry BxU199

10 15 20. 25 30 35 40 m

1

3.1

d

m m

1'01

1.001

FIGURE 4.-Kinetics of chromosomal transfer from U37cryB to U499.'

* Right scale: percent of gal+ recombinants.

18 V. ZAVADA AND E. CALEF

10 U37 cry 8 x U26L

1

- m m

01

001

FIGURE 5.-Kinetics of chromosomal transfer from U37cryB to U2M.'

* Right scale: percent of gal+ recombinants

insertion of such a long segment makes the gul-bio region too long to be encapsi- dated into a single P1 transducing particle (IKEDA and TOMIZAWA 1965). The cryptic prophage is clearly within the gul-bio region; it remains for P1 trans- duction experiments to decide definitely on which side of the inserted segment, but by analogy with the situation discussed by FISCHER-FANTUZZI (1967) its position is likely to be on the bio side. The segment inserted between gal and bio in both strains includes the locus purE as follows from preliminary mating experi- ments using a thr leu- pro- purE- guk female (ZAVADA, unpublished data). As both strains are lac- unlike their ancestor and the rest of its cryptic derivatives, it seems possible that the lac operon has been inactivated by the recombinational event leading to the transposition. Here, we should recall the data by BECKWITH and SIGNER (1966) and BERG and CURTIS (1967), where the transposition and inversion of the whole lac operon do not abolish its function.

Different kinds of chromosomal reorganization occurred with two other CO-

transduction-negative cryptic derivatives of U37 ( A crg) . With U37cryB, the polarity of chromosomal transfer was that of Hfr H with leu entering at 9 min and trp at 33 min. Both gal and bio have been transposed much nearer to leu as indicated by the respective entry times. Because of the extremely low frequencies of gal+ and bio+ progenies the precise times of entry of these markers and their mutual orientation on the chromosome cannot be estimated with enough accuracy

THE CHROMOSOME OF E. coli (Xcry)

TABLE 4

Genetic constitution of the gal+ progeny of crosses of U499 with U37 (A crg) and its cryptic deriuatiues.'

19

Donor leu

U37 ( A crg) +

U37cryQR A U37cryQ-R- B

U37cryQR- C

U3 7cryQ f R +

im - + + + + + + + + + + + + + + + + + - - - - + + + + + - - - - - + + + + + +

54 9 24 6 - . . . . . . . . . . . . . . . . . . . . . . . .

. .

. .

. .

. .

. .

. .

. .

. . +

. .

. .

. . . . . .

. .

. .

. .

. .

. .

. .

. .

. .

. .

. .

. .

Number

120

(d) = defective * Interrupted after 60 min of mating.

20

10

U1

K a K

- 1

n E

E

.-

0 V

-4- 0

0.1

0.01

V. ZAVADA A N D E. CALEF

U37 cry A X U199

P r””’ - rl

1 i gal

i

10 15 20 25 30 35 40 mi FIGURE 6.-Kinetics of chromosomal transfer from U37cryA to U499.

(see GLANSDORF 1967). The low frequencies of both types of progenies are pos- sibly due to the chromosomal rearrangement leading to anomalies in pairing. The decreased probability of integrating markers adjacent to exchange points in transposition strains has been reported by BERG and CURTIS (1967). Also, the exclusion of the bio allele of the male as well as of the alleles of the cryptic pro- phage from the gal+ progeny and vice versa (bio+ progeny carrying cryptic alleles) seems to be the consequence of the chromozomal rearrangement in this particular male.

So far we have no simple explanation also for the situation met with U37cryA. The early transfer of gal is obviously mediated by some sort of F-duction as follows from the sensitivity of the early gal+ progeny uersus f2. In addition, the cryptic male A is able to transfer its chromosomal markers with an Hfr H orien- tation at a reduced frequency.

So, while the males A and B enlarge the panorama of possible chromosomal

THE CHROMOSOME OF E. coli (hcry) 21

alterations in cryptic lysogens, they add little to our understanding of the forma- tion of the cryptic prophage. Perhaps males Q+R+ and C could give us a lead because both become lac- and both have the purE locus inserted between gal and bio. Tentatively they could be explained as the consequence of an inverted dupli- cation leading to an inversion. A detailed scheme for such an event has been given by C. THOMAS (THOMAS 1966; Figure 6). As a duplication has already been postulated for h cry (MARCHELLI et al. 19,68), the question is raised whether this duplication is the same and simultaneously responsible for both the prophage deletion and inversion. Our hypothesis can be presently formulated as follows: the prophage is capable of eliminating a portion of its genome because that portion is bracketed between two homologous sequences. After the deletion a new sequence is generated which is homologous and inverted with respect to a portion of the lac region; in turn this leads to the inversion of the segment containing the loci purE and gal and to the inactivation of the lac function by splitting that operon.

SUMMARY

The lack of PI mediated joint transduction of gal and bio is a frequent though not an obligatory phenomenon with E. coli strains carrying a cryptic h prophage between those loci. Chromosomal rearrangements affecting the gal-bio region in cotransduction-negative cryptic lysogens are not of a uniform type, as illus- trated by 4 independent cryptic Hfr H derivatives. In 2 of them the lack of gal- bio cotransduction is clearly due to insertion between the loci gal and bio of a chromosomal segment representing approximately 5 % of the total chromosomal length. The transposed segment is normally situated between leu and gal and contains the locus purE.

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APPLEYARD. R. K., 1954

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The formation of superinfection-double

Mutants osf Escherichia coli requiring methionine or vita-

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22 V. ZAVADA AND E. CALEF

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