TESTS OF RECIPROCALITY IN CROSSINGOVER IN PARTIALLY DIPLOID F’ STRAINS OF ESCHERICHIA COLZ

DOUGLAS E. BERG* AND JONATHAN A. GALLANT

Department of Genetics, Uniuersiiy of Washingion, Seattle, Washington 98105

Received August 6, 1970

ECOMBINATION in bacteria and bacteriophages is accomplished by break- age and reunion of DNA (MESELSON and WEIGLE 1961; KELLENBERGER,

ZICHICHI and WEIGLE 1961; MESELSON 1964; OPPENHEIM and RILEY 1966) often associated with DNA synthesis (STAHL and STAHL 1970), or, when very small distances are involved, by insertion of a single DNA strand into a recipient duplex (Fox and ALLEN 1964).

The intimate details of recombination by breakage and reunion are not yet understood. In E. coli, in particular, it is not clear whether recombination is usually reciprocal or nonreciprocal. In certain cases, the recovery of reciprocal products of exchange is evident: (1 ) the production of F’ variants in haploid Hfr strains through recombination between two regions of the Hfr chromosome (SCAIFE and PEKHOV 1964); (2) the production of haploid Hfr variants in F’ strains, through recombination between the F’ factor and the chromosome (BERG and CURTISS 1967; but see FAN 1969) ; (3) the production of tandem duplication Hfr strains, through recombination between homologous regions of the exogenote and endogenote (CUZIN and JACOB 1963; BERG and CURTIS 1967); (4) the in- tegration of phage lambda into the bacterial chromosome, through recombination between specific regions of phage and host (CAMPBELL 1962).

These examples show that recombination in E. coli is a t least sometimes re- ciprocal. However, the excision or integration of F’ factors or temperate phages demands the recovery of reciprocal products in order to be recognized at all. Thus, most of the situations cited above do not exclude the occurrence of non-reciprocal recombination, nor do they permit an assessment of its frequency, since non- reciprocal events would go undetected.

Another approach is represented by the experiments of HERMAN (1965. 1968) and MESELSON ( 1967), who examined F’ merodiploid recombinants in which the coupling relationships of markers had been altered. Both investigators found that reciprocally transposed classes (e.g., -+/+- from ++/--) were recovered in the same cell line with an appreciable frequency. In the case of MESELSON’S data, for example, 26% of the recombinant cell lines carried two reciprocally trans- posed genotypes.

These investigations confirm that recombination in E. coli is at least sometimes reciprocal, but again fail to exclude the occurrence of non-reciprocal recombina-

* Present address Department of Biochemistry, Stanford Umversity Medical Center, Stanford, California 94305 Requests for reprints should be sent to the Umvemty of Washmgton.

Genehcs 68: 467-472 August, 1971.

45 8 D. E. BERG A N D J. A. GALLANT

tion. A majority of the recombinant cell lines examined in both studies were, in fact, not reciprocal, but rather homozygous at one or more loci. It was not ascer- tained in either case whether homozygous recombinants arose non-reciprocally, or simply represented cases in which reciprocal products of exchange segregated to different daughter cells as in higher organisms (STERN 1936; PRITCHARD 1963).

This ambiguity is pointed out by two reports of non-reciprocality in the for- mation of homozygotes. GALLANT and SPOTTSWOOD (1965) and CURTIS (1968) stimulated the formation of homozygous recombinants through thymine starva- tion and ultraviolet irradiation, respectively, and examined the cell lines which were siblings of newly produced homozygous recessive recombinants. The re- ciprocal homozygous dominant products were not detected in either case. These results suggest that homozygous recombinants arise non-reciprocally. However, in both studies recombination was induced by lethal treatments, and the possi- bility remains that reciprocal products were formed but inactivated. A search for reciprocal products in the siblings of spontaneous homozygotes would certainly seem in order, and this is one of the purposes of the present investigation.

Taken all together, the investigations summarized above leave the question of reciprocal recombination in E. coli unresolved. The occurrence of reciprocal recombination has been decisively established, but it is by no means clear whether recombination is always reciprocal, as expected on the basis of simple exchange models, or only sometimes reciprocal, as expected on the basis of more refined models developed to explain the phenomenon of gene conversion. If a single basic mechanism underlies both reciprocal and non-reciprocal patterns, then it would be informative to know the relative frequencies with which recipro- cal and non-reciprocal events take place spontaneously. We have attempted such an accounting in the F’ partial diploid system.

We have employed both approaches described above. On the one hand, siblings of spontaneous homozygous recombinants were screened for the presence of reciprocally homozygous products. In order to frame a critical test of recipro- cality, it was necessary to distinguish between structurally different kinds of re- combinants, so as to remove from consideration events (see section 1.4 below) in which the proliferation of the reciprocal product, rather than its formation, might be at issue. This analysis, in agreement with MESELSON (1967), shows that the frequencies of different recombinant genotypes generates a “mitotic” map CO-

linear with the conventional map; this proves that recombinants issue from a process of exchange, rather than of localized gene conversion, and thus makes the whereabouts of the reciprocal exchange product a meaningful question. In most cases. we found that the production of one homozygous recombinant genotype was not accompanied by the production of a reciprocally homozygous sibling, suggesting that homozygote formation is rarely, if ever, truly reciprocal. These studies are described in section 1 of this report.

At the same time, we employed a method similar to that of Herman to detect reciprocally recombinant transpose genotypes in individual cell lines. Here again, reciprocality was found in only a small minority of recombinant clones. These experiments are described in section 2.

TESTS OF RECIPROCALITY 459

We are led to conclude that reciprocality in recombination is the exception, rather than the rule, in E. coli.

MATERIALS A N D METHODS

Bacteria: The bacteria used in this work are derivatives of Escherichia coli K12 (Table 1 ) . The structures of the Hfr strains, the F’ exogenotes, and the F’ partial diploids used in this work are shown in Figure 1.

Media: Minimal medium (GALLANT and STAPLETON 1963) was buffered with 0.1 M Tris, and supplemented with required amino acids, sugars, purines, and pyrimidines in optimal concentra- tions (LEDERBERG 1950). When specified, casein hydrolysate was added at 0.2%, streptomycin at 100 pg/ml, and triphenyl tetrazolium chloride at .0025%. When tetrazolium was used in minimal

TABLE 1

Bacterial strains

Hfr OR1 1 Hfr 13 DBL3 DB395 DB396 DB4.90 DB538 DB5M DB545 DB65/86

DB219/111

DB532/492

DB532/531

DBPD9

DBPD28

Hfr t hy leu; 0 proB . . . purE prd: lac F ; C. BERG and CURTIS, 1967 Hfr met 0 proC purE . . . proB lac F ; HIROTA and SNEATH, 1961 F- proC p u r E T p Zys met leu phoR lacZ,, str F’lac D34/Zac D34 thi; NEWTON et al., 1965 F’lac 06/lac 0 6 thi; NEWTON et al., 1 9 6 F- purE thy phoR lac p24 str F- purE phoR lacZ 0 6 F- purE proC phoR lac 06p24 F- purE proC lac 06 str F13pd lac Y Z phoA phoR T6 purE

F’ - + ts-i + s + thy

+ - a + - r - F’13pd lac phoA phoR T6 purE

F’ r? t s - i i + r + + + - s -

thy

F’ORF-8 lac phoR T6 purE pd F p24 f r +

0 6 - s - F’ORF-8 lac phoR T6 purE

thy ch str

pd F’ p24 $- + thychuurA6s tr 0 6 - s -

F’ORF-8 lac phoR T6 purE

pd - p24 + + t hychs t r f - s -

F‘ORF-8 lac phoA phoR T 6 purE

+ * + t hy pd F’ r7 -

+ + - s -

Abbreviations: The phenotype and genotype designations are as suggested by DEMEREC et al., 1966, except that T6 designates the locus of phage T 6 resistance, and ch designates uncharacter- ized auxotrophy satisfied by 0.1 % casein hydrolysate. pd designates partial diploid.

Particular alleles: pi-&, purE, tip, Zys, met, leu from x478 (C. BERG and CURTISS, 1967). uvrA6 from AB2437 (HOWARD-FLANDERS et al., 1966). lac06 (NEWTON et al., 1965). phoA ts-I and phoA -11 are inactive at 41”C, but active at 25°C. lacr7 reverts by mutation within the lac gene to a stable lac+ genotype with a high frequency. The bacterial strains used in this work were made by conjugation, segregation from partial diploids, and mutation.

460 D. E. BERG A N D J. A. GALLANT

lac

F',s PARTIAL DIPLOID ORF-8 PARTIAL DIPLOID

FIGURE 1.-Maps of haploid Hfr and F partial diploid strains. The upper panel shows the location of the lac-purE region on the chromosome of E. coli, and, in the enlarged arcs, the ap- proximate disposition of markers and F insertion sites in the Hfr and F' strains employed in these experiments. The lower panel shows the presumed structures of exogenote and endogenote when paired.

medium, lactose, casein hydrolysate and Penassay broth were added to I%, 0.2% and 10% final concentrations, respectively. For solid media, Difco Noble agar was added at 1 .e%. Complex media were L broth (LENNOX 1955), Penassay broth agar (Difco antibiotic medium No. 2), Penassay broth (Difco antibiotic medium No. 3), Difco MacConkey agar, and Difco Macconkey agar base supplemented with 1 yo of the appropriate sugar.

Mutant isoldon: Mutants were induced by treatment of cells with either N-methyl-"- Nitro-N-Nitroguanidine (ADELBERG, MANDEL and CHEN 1965) or with ultraviolet (W) light. Routinely 0.1% to 1% of the survivors of nitrosoguanidine treatment and less than .Ol% of the survivors of W irradiation were lac.

TESTS O F RECIPROCALITY 46 1

Quantitative conjugation: The donor and recipient cultures were grown in L broth at 37°C without aeration to a titer of 2-4 x 108/ml. They were mixed in the ratio of 1:9. After 1 hr of incubation at 37”C, the cells were agitated on a vortex mixer for 60 sec to disrupt chromosome transfer, diluted and plated on selective agar. The plates were counted after 18 to 60 hr incubation at 37°C depending on the growth rate of the recombinants.

Genotype determinations: On MacConkey agar lactose fermenting (lac+) colonies are red and nonfermenting (lac-) colonies are white; the reverse is obtained on tetrazolium lactose agar. Among lac- isolates, lacy+ were distinguished from lacy- at 41°C on MacConkey agar base SUP-

plemented with 1 % melibiose (BECKWITH 1964). Adenine (purE) sufficiency was determined on minimal medium supplemented with 0.2% casein hydrolysate and 10% Penassay broth. Since the purine level in this medium is limiting, purE- colonies are small, and purE+ are large. The genotypes at phoA and phoR were determined in the plate assay of GALLANT and SPOTTSWOOD (1965). For scoring phoA, the structural gene for alkaline phosphatase, colonies were grown at 41°C on minimal agar with the phosphate concentration lowered to 5 x 10-5 M in order to derepress the phoA gene. PhoR, the regulator gene, was scored in colonies grown on minimal agar with 2 x 10-3 M phosphate at 25°C. Resistance or sensitivity to phage T6 was determined by streaking broth cultures against T6.

UV stimulated recombination was used to reveal heterozygosity a t lac, phoA, phoR and purE. Cultures were streaked on the appropriate agar medium and irradiated with a Westinghouse germicidal lamp at 14 ergs/mmZ/sec for 20 to 30 sec. This test permitted the reliable detection of recessive alleles in heterozygotes without inducing significant numbers of forward mutants.

The structures of isolates were deduced from the frequencies of different classes of recombin- ants in conjugation experiments, and later from transfer patterns in a modified cross streak test (C. BERG and CURTIS 1967). Hfr 13 transfers proC efficiently and lac inefficiently, whereas F’ 13 strains transfer both the proC and lac regions efficiently. Most recombinants from F’ matings retain the lac region in partially diploid configuration. When a pr& Zac recipient is used Hfr and F’ donors can be distinguished by replica plating proC+ recombinants on MacConkey agar. The recombinants from Hfr 13 donors are Lac- in phenotype because they do not inherit the donor lac region. Recombinants from F’ matings are heterozygous or heteroallelic and ferment lactose.

Hfr OR1 1 transfers leu efficiently and proC inefficiently, whereas F’ ORF-8 strains transfer proC efficiently and leu inefficiently (C. BERG and CURTISS, 1967). It is possible to distinguish haploid Hfr recombinants from F’ partial diploid strains in cross streaks on the basis of proC vs. lac transfer (F’ 13) and proC vs. leu transfer (F’ ORF-8).

In the intragenic recombination experiments, lac heterozygous recombinants were character- ized to determine whether the lac+ allele was on the exogenote or on the endogenote, and to find which of the two mutant alleles was present in the lac- component of the heterozygote. Using a modified cross streak method, the isolates were crossed with a prof2 recipient carrying both lac- mutations (DB54-1.). proC+ recombinants were selected and replica plated to Mac- Conkey agar. Recombinants from F lac+/lac- donors ferment lactose strongly. Only weak fer- mentation was observed in recombinants from F‘ lac-/lac+ donors-and this probably resulted from rare transfer of the endogenote lac+ allele.

To determine which lac- allele was present in any heterozygous recombinant, several inde- pendent F’ lac-/lac homozygotes were selected from the heterozygote. They were cross streaked against purE- recipients carrying either lacp24 (DB538) or l ac06 (DB56), and the purEf recombinants were replica plated to MacConkey agar. The presence of interallelic lac+ recom- binants was used to indicate the genotype of the F’ donor.

RESULTS

1. Homozygous Recombinants ( 1 ) Structure of recombinants in F‘ 13 partial diploid strains: Heterozygous

partial diploid strains segregate cell lines which express a recessive allele. It is

462 D. E. BERG A N D J. A. GALLANT

TABLE 2

Transfer by Hfr 13 and by F’ 13 partial diploids

Average ratio Average efficiency Number of of proC+/trp+ Cotransfer of donor of proC transfer Genetic

Donor strain crosses transfer proC and lac alleles per donor structure __ DB219/111 4 409 110/120 0.23 F pa Heterozygous Lac+

revertants of Lac- segregants of DB219/111 5 595 160/176 0.65 F’ pd

Hfr 13 3 1.1 0/135 0.01 7 Hfr Haploid Lac+

revertants of Lac- segregants of DB219/111 5 2.1 0/190 0.027 Hfr

Crosses were performed with recipient DBL3 as described in MATERIALS AND METHODS. proC+ strr recombinants from each cross were picked directly to Macconkey agar without restreaking and incubated for 24 hr at 37°C. Those recombinants which fermented lactose were considered to have received the lac region of the donor and maintained it in a partially diploid condition. pro+ str* recombinants from the F’lac r7/lac+ donor (DB219/111) in the control cross accumu- late a small number of Lac+ intragenic recombinants during growth, which imparts a weak Lac+ phenotype to the entire colony when it is picked to MacConkey agar. Three of the five heterozy- gous revertants (Table, line 2) transferred the lac r7 allele, and thus were F’lac/lac+. The other two transferred the lac+ allele (the pro+ strr colonies fermented lactose strongly) and thus were FZac+/Zac. Each cross was performed with a different single colony isolate.

necessary to distinguish between homozygous and hemizygous segregants in order to ask whether the formation of homozygotes involves reciprocal exchange.

The most decisive criterion is ploidy, which can be established through a mutational test. Spontaneous Lac- segregants were obtained from a heterozygous F’ 13 Zac-/lac+ strain and characterized as to their pattern of reversion to Lac+. The diploidy of homozygous segregants was clearly revealed by the fact that their Lac+ revertants, being heterozygous, segregated Lac- subclones on restreak- ing. Reversion of haploid, hemizygous segregants, in contrast, generated stable lac+ clones.

A number of the Lac+ derivatives of these homozygous and hemizygous segre- gants was further characterized as to transfer pattern (Table 2).

The homozygous segregants all resemble the parental F’ 13 partial diploid strain: they transfer p r d at high efficiency, almost always jointly with lac, indicating F’ transfer; moreover, they transfer trp very much less efficiently, reflecting the low efficiency of chromosome mobilization. In short, the homozy- gous recombinants remain F’ partial diploid, as is to be expected. The hemizygous segregants, in contrast, resemble the ancestral Hfr 13 from which the F# 13 exogenote was originally derived; in particular, they never transfer lac as an unselected marker together with proC. This characteristic demonstrates that F has been reintegrated into the chromosome in between lac and proC, as in the ancestral Hfr 13. We will show below (Section 1.2) that the genotypes of these hemizygous Hfr segregants are consistent with a recombinational origin, although one which is inappropriate for tests of reciprocality. For the latter purpose, homo- zygous segregants were identified on the basis of their F’ rather than Hfr transfer pattern.

TESTS O F RECIPROCALITY 463

(2) Genetics of Lac- segregants from an F' 13 partial diploid strain: In order to analyze the genetics of segregant formation in F' 13 partial diploid strains, we constructed a strain heterozygous at six loci and obtained spontaneous Lac- segre- gants from a young broth culture. These were then characterized as to transfer pattern and genotype at all six loci (Table 3). It can be seen that 19% were F- segregants which had simply lost the exogenote, 12% were homozygous F' partial diploids, and 69 % were hemizygous Hfr recombinants.

The genotypes of the Hfr recombinants are grouped in Table 3 so as to display the hierarchical pattern into which the great majority distribute themselves. For any of the four markers to the right of F, inheritance of the endogenote allele is almost always accompanied by inheritance of endogenote alleles at every locus further from F; conversely, inheritance of the exogenote allele is almost always accompanied by inheritance of exogenote alleles for all loci closer to F. This kind of hierarchical array is typical of mitoltic crossing over in diploid organisms (PRITCHARD 1963; HOLLIDAY 1961). I t determines a unique linear order of the markers, along which crossover positions are reflected in the genotypes of the major recombinant classes.

All the major recombinant classes can be accounted for by a pair of exchanges bracketing the F factor, as illustrated in Figure 2. Table 3 lists the positions of exchanges corresponding to every recombinant class, including the rare quad- ruple exchange classes. The genetic map generated by these frequencies (Figure 3) is congruent with that established by conventional crosses (TAYLOR 1970). We conclude that the formation of hemizygous Hfr recombinants reflects an exchange process, rather than localized gene conversion.

The genotypes of the homozygous Lac- recombinants are similarly consistent with an exchange mechanism, as indicated in Table 3. They presumably arise

proC proC

lac

proC

F',s PARTIAL DIPLOID HAPLOID Hfr INVIABLE FRAGMENT

FIGURE 2.-Formation of haploid Hfr recombinants in F' 13 partial diploid strains.

464 D. E. BERG A N D J. A. GALLANT

TABLE 3

Genotypes of lac- segregants from an F'13 partial diploid strain

Locus lW phoA phoR T6 purE Crossover region 1 2 3 4 5 6 7

Genotype X = exogenote Y-Z+ F ts + r + N = endogenote Y+Z- - + - - S

crossover region lac Allele present

phoA phoR TL purE

B. Homozygous F' partial diploid 192 x/x 172 N/N

C. Infertile N X

Percent Percent Number of class of total

N X X X X N X X X X N N X X N N X X X

N N X X X N N X X X X X N N X N N N N

N N N N N N X N x x N N N N N N X N x x X N x x X N x x x x N X X N x x N X TOTAL:

N N

N N

X/N X/N X/N X/N TOTAL:

N N N N

TOTAL:

40 30 10

237 53 42 22

6 122 30 2 3 2 1 1 2 2 1 1

606 -

62 46

108 -

161 1

1 62 -

7 5 2

39 9 7 4 1

20 5

< I < 1 < I <I < I <1 < I < I < I 100 -

57 43

100 -

> 99 < 1 100 -

. .

. .

. .

. .

. .

. .

. .

. . 69

12

. .

19

Single colonies of the lac complementation heterozygote DB65/86 were picked from MacConkey agar, suspended in 1 ml of Penassay broth, grown to stationary phase, and plated on MacConkey agar. From those platings in which fewer than 10% of the colonies were L a c , several Lac- isolates were suspended in L broth, and cross streaked against DBL3 for pr&+ and unselected lac transfer in order to identify the segregants as haploid Hfr, homozygous F partial diploid, or infertile as described in MATERIALS AND METHODS. The genotypes of the segregants at phoA, phoR, T6, and purE were determined as described in MATERIALS AND METHODS. The genotype of DB65/86 is given at the top of the Table. The alleles present at each locus in the recombinants are reported in terms of their origin: X stands for the allele originally present on the exogenote, N for the allele originally present on the endogenote; in the case of diploids, X/N designates the parental heterozygous configuration, X/X designates homozygosity for the exogenote allele, N/N homozygosity for the endogenate allele.

TESTS O F RECIPROCALITY

purE

.59 -I- .I5 4

465

FIGURE 3.-Mitotic map of the F’13 exogenote. The map is based on the proportions of haploid Hfr recombinants listed in Table 3. For each arm on either side of F, the relative pro- portion of crossovers in each segment (Table 3) is the length of that segment relative to the overall length of the arm. The map shows these relative genetic distances. The two arms, of course, are not of equal length. A calculation based on the relative frequencies of Hfr and diploid homozygous recombinants (D. BERG, 1969a) suggests that the left arm is roughly half the length of the right arm, and the map is drawn to this scale.

when both exchanges occur on the lac side of F, and one exchange product then segregates with a nonrecombinant strand at cell division. Because F is located between lac and proC in the F’ 13 exogenote, such lac- homozygotes remain heterozygous at all marked loci on the proC side of F.

The two reciprocal types of homozygotes are recovered in roughly equal fre- quencies. The question we wish to ask, however, is whether they are produced jointly in single events. FJ 13 is clearly a rather inefficient system for posing the question, however, because of the low proportion of homozygotes among Lac- segregants. We therefore also examined Lac- recombinants in another partial diploid strain where homozygotes are more frequent.

(3) Genetics of lac- segregants from an F’ ORF-8 partial diploid strain: F‘ ORF-8 carries the same region of the chromosome as F’ 13, but F is located to the left of lac (BERG and CURTIS 1967) as shown in Figure 1. An F’ ORF-8 partial diploid strain heterozygous at four loci was constructed, and the genotypes of spontaneous Lac- segregants were determined (Table 4). It can be seen that more than half of the Lac- recombinants are homozygotes, presumably because of the more nearly terminal position of F in the ORF-8 exogenote. This proved advantageous in testing for reciprocality (section 1.4, below).

The Hfr recombinants show a linkage pattern in the recovery of exogenote alleles which is similar to that found in F’ 13. Among the homozygotes, a similar linkage pattern is seen in the retention of heterozygosity at the other three loci. This is a consequence of the fact that Zac and the other three loci are on the same side of F in this exogenote. We might emphasize that the genotypes of the homo- zygous recombinants, no less than those of the Hfr recombinants, conform to the expectations of a crossing-over model, and generate a “mitotic” map colinear with the conventional one. Table 4 indicates the positions of crossovers that will generate each recombinant type.

(4) Tests of reciprocality: Figure 2 shows that one crossover in the partially

466 D. E. BERG A N D J. A. GALLANT

TABLE 4

Genotypes of lac- segregants from an F’ORF-8 partial diploid strain

Locus F lac phoR T6 purE Crossover region 1 2 3 4 5 6

Genotype X = exogenote F - + + + N = endogenote - - - + S

Allele present Percent Crossover region lac phoR T6 purE Number of class

A. Haploid Hfr

B. Homozygous partial diploid 273 294 295 2,6 2,3,4,5 2,3,3,4 (4 strand) 2,3,3,4,4,5 (4 strand)

C. Infertile

N N N N X N X X X N N X

TOTAL:

X/N X/N X/N X/N X/X X/N x/x x/x x/x X/N X/N X/N

X/X X/N

TOTAL:

N N

14 4

47 12

1 1

79

42 2

43 7 1 1

1

97

2

-

-

18 5

59 15 1 1

43 2

44 7 1 1

1

I

Spontaneous Lac- segregants from DBPD28 were obtained as described in Table 3, and charac- terized with regard to transfer patterns and genotypes as described in MATERIALS AND METHODS. The genotype of DBPD28 is given at the top of the Table, and the terminology is as in Table 3. (The one exceptional homozygous recombinant which seems to represent a ghastly four strand exchange of rank six may reflect an error in classification.)

diploid regions on each side of F inserts F into the bacterial chromosome and generates a haploid Hfr structure. The reciprocal product, a small ring corre- sponding to the gene content of the F! exogenote, but deleted for F, has never been found. Two possible reasons are apparent: First, F probably supplies a part of the replication and segregation machinery for the exogenote (CUZIN and JACOB 1963; JACOB, BRENNER and CUZIN 1963). Consequently, exogenote frag- ments deleted for F are probably lost during cell growth, except under very special circumstances (see CURTIS 1964). Second, joint recovery of both the Hfr chromosome and the fragment would not be possible if either crossover were nonreciprocal. As a consequence of the first consideration, haploid Hfr recombi- nants must be excluded from consideration in tests of reciprocality.

The reciprocality of recombination can be tested quantitatively in two ways in the F’ partial diploid system: Following a pair of reciprocal crossovers, both

TESTS O F RECIPROCALITY

TABLE 5

Fermenting fraction of colonies sectored for Lac- homozygotes

46 7

Number of colonies without reciprocal

Colonies with reciprocal Lac+ homozygotes

Partial diploid Lac+ homozygotes Number Fraction that were reciprocal

F‘13 ORF-8

15 63

4 12/12; 2/12; 1/12; 1/12 7 10/12; 2/12; 2/12; 1/12;

1/12; 1/12; 1/16

Sectoring colonies of F’13 partial diploid DB219/11 and F’ORF-8 partial diploid DBPDQ were suspended in broth, plated on Macconkey agar, and 12 to 16 fermenting isolates from each popu- lation were tested for heterozygosity at lac (see MATERIALS AND METHODS). Colonies in which fewer than half of the cells were Lac-, or in which fewer than half of the Lac- isolates were diploid were not used. Partial diploidy was determined by transfer patterns in cross streaks or by heterozygosity at purE (see MATERIALS AND METHODS).

on the same side of F, the two crossover products sometimes segregate together generating a heterozygous recombinant. These are scored in section 2.3 below. Products of recombination also segregate with nonrecombinant homologs at cell division, and generate homozygotes. If this were a reciprocal process, the cell in which recombination takes place would give rise to two subclones of reciprocally homozygous genotype.

The reciprocality of spontaneous homozygote formation was tested by deter- mining whether colonies with large Lac- homozygous sectors contained a com- parable number of cells of the reciprocal Lac+ homozygous genotype. Cells from the fermenting (Lac+ heterozygous or homozygous) half of sectored colonies were isolated and their genotypes determined (Table 5). In 79% of F’ 13 partial diploid clones and in 89% of ORF-8 partial diploid clones the reciprocal class of Lac+ homozygotes was not found. Lac+ homozygotes comprised less than 20% of all fermenting isolates in nine of those eleven clones which did contain recipro- cal genotypes. This low frequency suggests that the two homozygous genotypes in these clones arose from several, rather than single events. Lac- and Lac+ homo- zygous genotypes were present in equivalent proportions in only two of the 85 clones examined. These results are not due to any normal selection pressure, since reconstruction experiments (D. BERG 1969a) show that Lac+ homozygotes are not less fit than other genotypes. Thus, spontaneous homozygote formation is rarely, if ever, a reciprocal process.

2. tntragenic Recombinants (1 ) Design: The second test of reciprocality determines the frequency with

which the partners in recombination segregate together, forming recombinant but heterozygous progeny. In this test, Lac+ intragenic recombinants were selected; note that their recovery does not depend on any particular segregation pattern, only the occurrence of an exchange between the sites of the two mutant alleles which are in repulsion. Reciprocality in intragenic crossing over is indi- cated by the occurrence of heterozygous recombinants carrying the two mutant alleles in coupling.

468 D. E. BERG A N D J. A. GALLANT

TABLE 6

Intragenic Lac+ recombinants from heterwllelic F'ORF-8 partial diploids

Genotype uvrA6 uvrA6' To ta l +

A. lac phoR No. % No. 2 No. %

+/N X/N 66 37 38 23 104 30 x/+ X I N 31 18 29 17 60 1 7 +/X X I N 14 8 14 8 28 8

XI+ xlx 19 11 33 20 52 15 +lX xlx 13 8 11 7 24 7

- -

N/+ X/N 3 2 3 2 7 2

1

+lx NIX 4 2 3 2 NI+ NIX 5 2 8 5 XI+ N I X 2 1 1 <1 +IN N I X 3 2 6 4 +/XN N I X 1 <1 0 0 XN/+ NIX 0 0 6 4

7 2 1 3 4 3 <I 9 3 1 <I 6 2

I I +/XN X/N NI+ XIX TIN xlx +/x N/N N/+ N/N

B. lac allele - present

+,N +,x +,m

C. Tota l

11 6 1 <1 2 1 1 <1 1 <1

81 46 84 47 1 2 7

17 7

7 1 2 1 2

6 1 92 13

166

4 <1 1 <1 1

37 55 8

18 5 2 <1 4 1 2 <1 3 <1

Lac+ intragenic recombinants accumulating during the growth of DB532/492 (uurA+) and DB532/531 (uurA6) on Macconkey agar were selected, purified by restreaking once, and then tested for genotype as described in MATERI~LS AND METHODS. 90% were F' partial diploid and heterozygous and are summarized in the Table. The genotype designation indicates the allele present, and its position. In the appropriate columns X designates lacp24 or phoR+ (parental exogenote alleles), and N designates lac 0 6 o r phoR- (parental endogenote alleles). The genotype of the recombinants is presented as: exogenote/endogenote Thus, the lac genotype f / N designates F'lac+/Zac 06, and the lac genotype XN/+ designates F'lac p24,06 (cis double mutant) /Zac+.

TESTS O F RECIPROCALITY 469

The recovery of such reciprocal recombinants also depends on the joint segre- gation of both recombinant homologs to the same daughter cell. The segregation pattern was monitored by constructing partial diploid strains heterozygous for the linked p h R locus: the exogenote was phoR+ and the endogenote was phoR-. The usefulness of the linked phoR marker will become apparent in section 2.3 below.

QRF-8 F” partial diploid strains were employed because of the linkage of lac and phoR. This permits detection of those events in which crossing over within lac actually resulted in the exchange of a large block of genetic material, and also because partially diploid recombinants arise more frequently than haploid recom- binants in this strain (section 1.3, above).

( 2 ) Rate of recombination: The strains described above generated about 0.2% Lac+ recombinants in the course of 30 generations of growth. The rate of recom- binant formation per cell per generation is thus 2 x 10-3/30 or about 7 X Similar results were obtained with both uurA+ and uurA- diploids, indicating that the uurA gene does not affect the probability of spontaneous recombination.

The exogenote lac2 point mutant was crossed with lac2 deletions in order to locate its position. Lac+ recombinants were obtained with deletion 015, but not with deletion D34, which places the point mutant in the one eighth segment of the lac2 gene adjacent to lacy (NEWTON et al. 1965). The endogenote lac muta- tion in our diploids was lacO6, a deletion of the operator proximal one fourth of the lac2 gene (NEWTON et al. 1965). Thus, about 60% of the lac2 gene, or 2200 base pairs, separate the two mutations in the heteroallelic diploids, a distance within which crossing over occurs with a probability of 7 x per cell per generation. Thus, there is one crossover per 3 x lo8 base pairs per generation in these F’ partial diploids.

(3) Genotypes of lac+ intragenic recombinants: Table 6 presents the geno- types of Lac+ recombinants in uvrA+ diploids. The uvrA allele had little effect on the pattern observed. If crossing over were at a four strand stage, and the segregation of homologs to daughter cells were at random, as in higher forms, then half of the Lac+ cells should carry both the homologs that underwent ex- change. Were recombination reciprocal, then this 50% would carry the cis double mutant. On the contrary, only about 8% of the Lac+ recombinants were of this genotype.

The test of reciprocality would be most critical among Lac+ recombinants in which we had reason to believe that crossover products had segregated together. In those Lac+ recombinants in which the adjacent phoR locus was included in the region of exchange, the exagenote and endogenote phoR alleles would ex- change places: the exogenote R+ allele would be shifted to the chromosome, while the endogenote R- allele would be shifted to the episome. When these exchange partners segregate together, the resulting Lac+ recombinant will thus carry the phoR alleles in transposed configuration. This class of Lac+ recombinants is bracketed in Table 6. I t can be seen that only 7 out of 39, o r 18%, carry the reciprocal cis double mutant.

DISCUSSION

In F’ partial diploid strains estimates of the extent of reciprocality are compli-

470 D. E. BERG A N D J. A. GALLANT

cated because some types of recombinant molecules are probably inviable. Conse- quently, analyses of reciprocality should be preceded by an examination of chro- mosome behavior in the partially diploid F’ strains so that criteria can be estab- lished for distinguishing homozygous recombinants from haploid Hfr recom- binants.

Haploid Hfr recombinants and homozygous recombinants are both formed through a pair of exchanges in the partially diploid segment. A haploid Hfr chromosome (and probably an inviable fragment as well) is generated by in- sertion of F into the bacterial chromosome when one crossover occurs on each side of the F factor. When the crossovers are restricted to one side of F, homozy- gotes are formed provided that a recombinant element segregates with a nonre- combinant homolog at cell division. Analysis of haploid Hfr and homozygous recombinants generates genetic maps that are congruent with the maps generated from conjugation and transduction data.

Single events of homozygote formation, if caused by reciprocal crossovers, should result in the correlated production of two reciprocally homozygous geno- types. However, reciprocally homozygous genotypes were recovered in only 1 1 of the 85 clones examined. Because recombination events are clustered within a clone, rather than independent (D. BERG 1969a,b), it is likely that at least some of the apparently reciprocal clones are the fortuitous result of several nonrecipro- cal events. Thus, 11/85 (13%) is probably an upper estimate of the frequency of reciprocal events. Our inability to recover reciprocally homozygous genotypes in most clones makes it appear that spontaneous homozygote formation in partially diploid F’ strains is normally a nonreciprocal process.

One alternative possibility may be considered. If exchange occurs when only one of the homologs is duplicated, then at subsequent cell division one daughter cell would be homozygous and the other heterozygous, just as we observe in most cases. In order to control for this eventuality, as well as to complement the previ- ous approach, we also sought the elusive reciprocal product among Lac+ intra- genic recombinants segregating from strains which were heteroallelic at 1ac.Z.

Here again, the reciprocal product, the cis double mutant, was encountered only rarely. Under the assumption of crossing over at a four strand stage and random segregation, 50% of the Lac+ recombinants would be expected to carry both products of exchange.* Thus, the 8% reciprocals we found among the gener- al Lac+ recombinant population might reflect 16 % reciprocal recombination events.

In close agreement with this figure. we found 18% (7/39) reciprocal types among Lac+ individuals whose transposed genotype at phoR implied the joint segregation of both crossover products. We might note that these cells apparently contain reciprocal products of exchange as far as the phoR region is concerned. Yet even among these cells, reciprocality close to the crossover site (in lac) is exceptional. This observation suggests that the probability of recovering genetic

* If crossing over were at a three strand stage, followed by replication of the unreplicated homolog, ’2/3 of the Lac+ recombinants would carry the double mutant. If crossing over were at the two strand stage, all of the Lac+ recombinants would carry the reciprocal product. In other words, 50% is the minimum to be expected if segregation is random.

TESTS O F RECIPROCALITY 471

material from the (presumed) reciprocal product of exchange increases with distance from the site of exchange, in agreement with HERMAN’S (1968) con- clusions. Nevertheless, the rarity of reciprocal homozygotes shows that non- reciprocal behavior is still the rule at considerable distances (several cistron lengths at least) from the site of exchange.

The simplest interpretation is that recombination in F’ partial diploid strains is basically a nonreciprocal process. but that “reciprocal” products can be pro- duced infrequently as a result of compound events. These outcomes support models in which nonreciprocal crossing over occurs between already replicated homologs (STAHL 1969) or in which crossing over itself generates an unusual replication structure and a 2:l ratio of alternative alleles (BOON and ZINDER 1969). On both of these models, “reciprocal” recombinants would arise following a more complicated and therefore less frequent sequence of events, just as we have observed.

This work was supported by PHS Training Grant GMOOl82 and by grant number GM13626 from the National Institutes of Health.

S U M M A R Y

Crossingover occurs at a low rate in established partially diploid F’ strains of Escherichia coli, and generates haploid Hfr, homozygous F# partial diploid, and heterozygous (recombinant) F’ partial diploid recombinant strains. We find that homozygotes comprise the major class of recombinants; they do not arise recipro- cally. Additional nonreciprocal behavior, limited to the crossover region, is also evident, suggesting that the recombination process in E. coli is rarely, if ever, truly reciprocal.

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