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Reciprocality of Recombination Events That Rearrange the Chromosome Michael J. Mahan and JohnR. Roth Department of Biology, University of Utah, Salt Lake City, Utah 841 12 Manuscript received January 6, 1988

Revised copy accepted May 16, 1988 ABSTRACT We describe a genetic system for studying the reciprocality of chromosomal recombination; all substrates and recombination functions involved are provided exclusivelyby the bacterial chromosome. The genetic system allows the recovery of both recombinant productsfrom a single recombination event. The system was used to demonstrate the full reciprocality of three different types of recombination events: (1) intrachromosomal recombination between direct repeats, causing deletions; (2) intrachromosomal recombination between inverse homologies, causing inversion of a segment of the bacterial chromosome; and (3) circle to circle recombination (in the absence of any plasmid or phage functions). Results suggestthat intrachromosomal recombinationin bacteria is frequently fully reciprocal.

T is fundamental to our understanding of recombinational mechanisms to know whether or not an exchange is fully reciprocal (resulting in rejoining both pairs of flanking sequences) (CLARK1973; MESELSON and RADDING 1975; STAHL1979a,b; RADDING 1982; SMITH1983; SZOSTAK et al. 1983). Reciprocality has been shown to be characteristic of recombination in many situations. In fungi, genetic analysis is facilitated by the recovery of all products of a single meiotic event. This property of fungal systems has provided ample evidence that recombination in these organisms can be fully reciprocal. In bacterial systems it is more difficult to determine whether recombination is reciprocal since the products of a single event are generally unassociated. In the tests described here, we are most interested in distinguishing between fully reciprocal and “half-reciprocal” exchanges (Figure 1). This distinction is particularly important when one considers genome rearrangements generatedby recombination between homologies located at separated chromosomal sites. Fully reciprocal exchanges are formally required for exchanges within a single chromosome that form an inversion and for events that integrate onecircle into another, while duplications and deletions can be generated by either full- or half-reciprocal events between sister chromosomes. Previously, phage lambda crosses and marked lambda double lysogens have been used to study the reciprocality ofrecombination in bacteria. A statistical analysis was performed on the recombinant progeny derived from genetic crosses involving multiply marked lambda chromosomes present either in doublelysogens (MESELSON 1967), or in lambdalytic crosses (SARTHY and MESELSON 1976). The results from both types of crosses yielded reciprocal recom-

I

Genetics 120: 23-35 (September, 1988)

bination types at equal frequencies even insingle bursts, leading tothe conclusion thatunder these conditions recombination is fully reciprocal in bacteria. While these results are convincing, our interest lies in the recombination events involved in various chromosome rearrangements. We would like to distinguish between fullyreciprocal and half-reciprocal exchanges in an effort to understand the processes by which these rearrangements occur. Another strategy to test the reciprocality of chromosomal recombination in bacteria is the integration and excision of F’ episomes. CAMPBELL (1962) proposed that F’ episomal factors integrate into the bacterial chromosome by a single fully reciprocal crossover resulting in the formation of an Hfr strain with a duplication of the sequences at the site of integration. The reversal ofthisprocess regenerates the original F’ episome (CAMPBELL 1962). The results of these typesof experiments suggested that rec-mediated (host encoded) recombination was fully reciprocal in bacteria (HERMAN 1965, 1968). Circle to circle recombination certainly implies full reciprocality, because both products from a single recombination event are recovered. However, replication and recombination functions encoded by the plasmid may have contributed to theevents observed. Thus, there are inherent complications in testing reciprocality ofbacterial recombination. (1) The products of one event are not usually held together. (2) Most test systems perturb the cellinsomeway,by subjecting it to infection, conjugation, or transduction and by introducing foreign functions that may contributetothe course of the event. (3) Many test systems present substrates that may not normally be seenin a cellsuchas double strand ends, atypical supercoiling, or single stranded substrates.

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M. J . Mahan and J . R. Roth

rullY-gertrrcnrp1 A

a

a

B

X

\

A

a

b

b

A

b

a

B

A

b

a

B

_I__)

-

B

/

b

__I)

FIGURE1.-Reciprocal recombination. Fully reciprocal recombination demands the joining of both pairs of flanking markers from a single exchange. Half-reciprocal recombination demands the joining of only one pair of flanking markers from a single exchange.

This paper addresses someof these problems through use ofa genetic system in which allsubstrates and recombination functions are provided by the bacterial chromosome. The cells are not perturbed by infection or the presence of a foreign replicon. The system tests the full reciprocality of three different types of recombination events: (1) intrachromosomal recombination between direct repeats, causing deletions; (2) intrachromosomal recombination between inverse repeats, causing inversions; and (3) circle to circle recombination (with no involvement of plasmid or phage recombination functions). The genetic test also allows one to assess the contributions of interand intrachromosomal exchanges to segregation of a small duplication. MATERIALS AND METHODS Bacterial strains: All strains used in this study (Table 1) were derived from Salmonella typhimurium LT2. All directed transposition strains were constructed according to methods described previously (CHUMLEYand ROTH 1980)and (SCHMIDand ROTH 1980). Deletion mutation cob21* MudJ*hisF9951 (TT11077), which removes approximately 50 kb of material, including part of the his operon, was constructed as described previously (HUGHESand ROTH 1985). Isogenic strains carrying deletion mutations hisOGD646 or hisOG203 were constructed by transduction. P22 phage grown on strain TT11829 (duplication from the trp region clockwise to hisH with TnlO at thejoin point) was used to

transduce strain hisOGD646 to tetracycline resistance (Tc') (this requires inheritance of the duplication). Twenty percent (20 of 100) of the Tc' transductants were his auxotrophs, indicating that h i s 0 0 6 4 6 was present in both copies of the inherited duplication. P22 phage was grown on one of the His- Tc' transductants (TT1 183 1) and used to transduce LT2 totetracycline resistance. Five Tc' His+ transductants were isolated. The prototrophic transductants were grown overnight in liquid nutrient broth medium nonselectively in order toallow loss of the wild type his copy, leaving a segregant clone with a haploid copy of hisOGD646 that had been introduced into anLT2 background (TT11834). The same procedure was used to introduce hisOG203 into the LT2 genetic background (TTll836). Isolation of His+ recombinants and Hol- segregants: Strains from a frozen culture of the parent strain to be tested (see RESULTS for details) were streaked for single colonies on nutrient broth solid medium and grown 20-22 hr at37'. One colony was resuspended in 1 ml of E medium. This suspension was split into three fractions; one fraction was used for each of the following determinations. The number of cellgenerations was calculated ( N = N0F, where x is the number of cell generations, NOis the number of cells at time zero, and N is the number of cells (viable and inviable) at time t; since the resuspended colony arose from a single cell, No = 1). N was determined by counting the number of cell particles in suspension using a Coulter Counter (model F, 30 pm orifice, Coulter Electronics). Pop1 ulations tested further (see below) had undergone 24 generations in forming the colony from the single plated parental cell. In order to determine the Hol- segregation frequency, serial dilutions of fraction 2 were plated for single colonies (ca. 200/plate) nonselectively on solid nutrient broth medium and incubated overnight at 37". The colonies were then replica printedto (1) minimal medium containing histidinol (Hol) and (2) minimal medium containing histidine in order to score the loss of the hisD chromosomal segment (Hol- segregant). The frequency of Hol- segregants was calculated as the number of Hol- segregants per viable cell plated. His+ recombinants were selected on minimal medium. Since coloniescontinue to appearwith extended incubation times, 38 hr of incubation at 37' was arbitrarily chosen for assaying colonynumber. The number of coloniesarising on the plate was found to be dependent on theextent of residual growth on the selection medium and thus on the residual nutrients plated. T o makethis residual growth uniform, cells were plated on a lawn of a strain containing a his deletion (hisO-E3050) which consumes the plated nutrients. Under these conditions, the number of His+ recombinants detected at 38 hr of incubation was linear with the number of parental cells plated. The frequency of His+ recombinants throughoutthe paper is expressed as the number of His+recombinant colonies scored per viable cell plated. Genetic assay for the presence of his sequences at the nru locus: Strain TTllO77, containing a large his deletion associated with a Km' determinant (cob21*MudJ*hisF9951), was constructed according to HUGHESand ROTH (1985). P22 phage grown on this deletion mutant was used to transduce the His+ derivative of the parent strain to kanamycin resistance (Km'). Since the deletion removes all the his material from the hisF gene through the his promoter into thedistant cob genes, the only hisD sequences remaining in transductants that inherit the deletion must be located at the ara region (see RESULTS for complete description). Therefore, the state of the his sequences at ara is revealed

*

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Reciprocality of Genome Rearrangements TABLE 1 Bacterial strains Strain

Genotype

Method“

Recipient

hisOGD646 proAB47 his135 hisOG203

Donor

TR6535 TR6976 T T 5 13 TT1151 TT1704 TT3003 T T 3 164 TT3291 TT3699 T T 6 2 12 TT7701 TT8057 TT10286 TTllO77 TT11782 TTll783 TTll785 TTll786 TTll787 TTll788 TT11789 TTll790 TTll791 TTll792 TTll793

hisOGD646, proAB47 his135, strA1 zee-2:TnlO(A)’ hisC8691::TnlO(A)’ hisO-E9533 hisOGD646, hisC8691::TnlO(A)’ zee-a::TnlO, hisC129 zee-2::TnlO, his01242-H8698:Tn10 ara-651::TnlO(B)’ pncB165::TnlO(A)’ hisH9962::MudA pncB22O::MudA hisD9953::MudJ DeI646[(cob21)*MudJ*(hisF9951)] ly~J8l::MudF zee-P::TnlO(A)*, hisC8691::TnlO(A)’ ara-651::TnlO(B)’, his9533 pncB165::TnlO(A)’, his9533 ara-651::(TnlO-hisO-C8691-TnlO), his9533 pncB165::(Tn10-his0-C8691-TnlO), his9533 ara-65l::(TnlO-his01242-H8698-TnlO), his9533 pncBl65::(TnlO-hisO1242-H8698-Tn10), his9533 ara-65l::(TnlO-hisO-C869l-TnlO), hisOGD646, proAB47 pncBl65:;(TnlO-his0-C869l-TnlO), hisOGD646, proAB47 ara-65l::(TnlO-hisO-C8691-TnlO), DUP764[(hisOGD646C8691)*TnlO*(hisO-E+)],proAB47

T T T T

TTll797

INV768[(ara-651::TnlO)*his+*(hisC869l::TnIO-his-

His+ revertant of TT 1 179 1

TT11830 TT11831 TTll832 TTll833

TT 11834 TTll835 TTll836 TTll841 TT11842 TT11843 TTll844 TT12914 TT13788 TT13789

TT13790

TT13791 TT13792 TT13793

OGD646)], zee-2:TnlO,proAB47 DUP782[(zde-1874 - hisH8546)*TnlO*(trp+ his+)] DUP782[(zde-1874 hisOGD646-hisH8546)*TnlO*(trp+ hisOGD646)l DUP782[(zde-1874 - hisOG203”hisH8546)*TnlO*(trp+ hisOG203)I DUP782[(zde-1874 - hisOGD646-hisH8546)*TnlO*(trp+his+)] hisOGD646 DUP782[(zde-l874 - hisOG203-hisH8546)*TnlO*(trp+ his+)] hisOG203 pncB165::(TnlO-his0-C869I”nlO),hisOGD646 pncB165::(TnlO-hisO-C869l-TnlO), hisOG203 pncBl65::(TnlO-hisO1242-H8698-TnlO), hisOGD646 pncB165:(TnlO-hisO1242-H8698-TnlO), hisOG203 pyrC7, Str‘, F’114 ts lac+ zzf1836::TnlOd-Cam ara-651::(TnlO-his0-C869I”nlO),proAB47, cob-jrO::MudA, F‘114 ts lac+ zrf-1836::TnlOd-Cam ara-65l::(TnlO-hisO-C869l-TnlO), DUP764[(hisOGD646C8691)*TnlO*(hisO-E+)],proAB47, cob-3O::MudA, F’114 ts lac+ zrf-1836::TnlOd-Cam INV768[(ara-651::TnlO)*his+*(hisC8691::TnlO-hisOGD646)], proAB47,cob-30::MudA, zee-P::TnlO, F’114 ts lac’ zrf-1836::TnlOd-Cam ara-651::(TnlO-hisO-C8691-TnlO), proAB47, leuAll79::MudA, F’114 ts lac+zzf1836::TnlOd-Cam ara-651::(TnlO-hisO-C8691-TnlO), DUP764[(hisOGD646C8691)*TnlO*(hisO-E+)],proAB47, leuAl179::MudA, F’114 ts lac+ zrf-1836::TnlOd-Cam INV768[(ara-651::TnlO)*his+*(hisC8691::TnlO-hisOGD646)], tee-a::TnlO, leuAl179:MudA, proAB47, zee2:TnlO, F’114 ts lac+ zrf-1836::TnlOd-Cam

-

-

-

TR5667 Pooled TnlO insertion 1t2 Pooled TnlO insertion TnlO generated HisG- from zee-1::TnlO T hisOGD646 TT 1151 T hisC129 13 TT5 (SCHMID and ROTH 1980) T 1t2 Pooled TnlO insertion T 1t2 Pooled TnlO insertion T 1t2 TT7688 X TT7610 T 1t2 TT7688 X TT7610 T 1t2 TT10270 X TT7692 T 1t2 EH425 X TT10687 T TT12116 TT10377 T TT T3T030 3 164 T TT 1704 TT3699 T TTT l 76024 12 T TT11783 TT11785 T TT11783 TT11786 T TTTT3l2l 7 98 15 T TT3291 TT11786 T TR6535 TTll787 T TR6535 1788 TT 1 His+ revertant of TT 1I79 1

T T

hisOGD646

1t2

TTll853 TTll830

T

hisOG203

TTll830

T

1t2

TT11831

Tc” His- segregant of T T l l 8 3 3 T 1t2 TTll82 Tc’ His- segregant of T T l l 8 3 5 T TT11788 TT11834 T T TT Tllll788386 T T T l l 78 93 04 T TT11790 TT11790 T TT10604 TR2647 C TT12914 TT13652 C

TT12914 TT13653

C

TT12914 TT13654

C

TT13655

TT12914

C

TT13656

TT12914

C

TT12914 TT13657

~

a

The method of construction is indicated. “T”denotes P22 mediated transduction. “C” denotes conjugation. TnlO (A) refers to a TnlO insertion in orientation A; TnlO (B) refers to a TnlO insertion in orientation B.



M. J. Mahan and J. R. Roth

26

by scoring the ability of the Km’ transductants (which must Parental Strain inherit the deletion) to utilize histidinol (Hol) as a histidine (TT11791) source. Ten Km’ transductants derived from each His+ recombinant were scored for ability to grow on histidinol (HisD+).Growth on histidinol demonstrates the presence of a hisD gene, inferred to be at the ara locus. (Genetic evidence confirming that the location of the hisD gene is at the ara locus is presented in the APPENDIX). Nomenclature: Nomenclature is generally as described et al. (1966), CAMPBELL et al. (1977), and in DEMEREC CHUMLEY, MENZELand ROTH(19’79). The nomenclature z--::TnlO refers toaTnlO insertion in a “silent” DNA region; the “z--” describes the map position of the insertion (SANDERSON and ROTH 1983). The nomenclature used for chromosomal rearrangements is described in CHUMLEY and ROTH (1 980), SCHMID and ROTH(1983), and more recently in HUGHES and ROTH(1985). Media: The E medium of V ~ C E L and BONNER(1956) supplemented with 0.2% glucose was used as the defined minimal medium. Selection for growth on alternative carbon sources was done on NCE medium, described by BERKOWITZ et al. (1968), supplemented with 0.2% of the appropriate carbon source. The complex medium was nutrient broth (8 g/liter, Difco Laboratories) with added NaCl (5 g/ liter). Solid medium contained Difco agar at1.5% final concentration. Auxotrophic requirements were included in media at final concentrations described by DAVIS, BOTSTEIN, and ROTH(1980). Final concentrations of antibiotics were: FIGURE2.-The parental strain used in the reciprocal recombination assay. The ara region (TTI 1791 ) contains a hisOGDC’ tetracycline hydrochloride (Sigma Chemical Co., 16 pg/ml in rich medium, or 10 pg/ml in minimal medium); kanachromosomal segment flanked by two TnfO elements (represented by solid arrows) in the same orientation (C’ indicates that only part mycin sulfate (Sigma Chemical Co., 50 pg/ml in rich medium, or 100 pg/ml in minimal medium); ampicillin (Sigma of the hisC gene is present here). The parent strain also contains a deletion in its standard his region (hisOGD646). The his sequences Chemical Co., 40 pg/ml in rich medium, or 15 pg/ml in minimal medium), chloramphenicol (Sigma Chemical Co., are placed at ara in inverse order with respect to the orientation of the normal his operon. 20 pg/ml in rich medium or 5 pg/ml in minimal medium); 6-amino-nicotinicacid (Sigma Chemical Co., 50 pg/ml minOne of the parent strains used in this test imal medium). Transductional methods: The high frequency general(TT11791) is diagrammed in Figure 2. The ara reized transducing bacteriophage P22 mutant H T 105/1, intgion contains the proximal portion of the his operon 201 (SCHMIEGER 1972) was used for all transductional flanked by two TnlO elements in the same orientation; crosses. Unless otherwise specified, 0.1 ml of an overnight the his material present at ara is in inverseorientation culture grown incomplexmedia (ca. 2-4 X lo9 colonyvis (I vis the standard his operon. The presence of his forming units/ml) was used as a recipient of 0.1 ml transducing phage (ca. 108-109 plaque-forming units/ml) and material at ara may be scored genetically due to the plated directly on selective plates. Transductional crosses presence of a functional hisD gene. The parent strain involving the selection of kanamycin or chloramphenicol carries a deletion in its standard his region that reresistance were preincubated overnight on solid nonselecmoves the his promoter and the proximal portion of tive complex medium, then replica printed onto selective the his operon, including part of the hisD gene. The medium. Transductants were purified and phage-free isolates were obtained by streaking for single colonieson green phenotype of the parentstrain is His- sinceit lacks an indicator plates (CHANet al. 1972). Phage-free colonies were expressed hisC gene. The strain is able to grow on tested for phage sensitivity by cross streaking with P22 H5 histidinol as a histidine source (Hal+) due to expres(a clear plaque mutant of phage P22). RESULTS

Assay for reciprocality:The genetic test described here detects both recombinant products of a reciprocal exchange between homologous sequences in the same chromosome. A fully reciprocal exchange between direct order homologies can yield a chromosomal deletion and an extrachromosomal circle. A reciprocal exchange event between inverse order homologies on the same chromosome yields an inversion. Both types of recombination events have been identified and are discussed below

sion of the hisD gene in the chromosomal segment located at ara. Events that can be detected inthis strain are described below. Deletion formation and circle generation: The test for the reciprocality ofrecombination between direct repeats involves the loss of his sequences from ara, generating a circle, and the recaptureof this circle. If intrachromosomal recombination between direct repeats is fully reciprocal, an exchange between the two TnlO elements (top of Figure 3) would generate two recombinant products: (1) an excised circle of DNA containing the hisOGDC’ chromosomal fragment and a TnlO element; and (2)a restored parental chromo-

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Reciprocality of Genome Rearrangements

No hom0lOgy

with clrcle

+

captured ctrcle a t RfS

broken clrcle

intact chromosome

(Case 2)

-++

FIGURE4.-Recapture of the circular product. Circles generated by either fully reciprocal or half-reciprocal intrachromosomal recombination may be recaptured at thehis operon. A circle to circle recombination event involving the hisDC homology of the free circle and thehisDC homology of the chromosome generates a His+ recombinant. A disrupted circle (linear) cannot repair hisOGD646 since there is no homology to the left of the deletion for homologous recombination. The dark arrows represent TnlO elements in the same orientation.

intact clrcle

broken chromosome

FIGURE3.--Generation of free excised circles. Circles may be generated by either fully reciprocal or half-reciprocal recombination. Fully reciprocal intrachromosomal recombination between direct repeatsat ura generates (1) a free excised circle of DNA; and (2) a concomitant restoration of the donor chromosome that is associated with a deletion of his material from the donorsite. Halfreciprocal intrachromosomal recombination between direct repeats at the donor site generates only one recombinant product, either (a) an intact chromosome associated with a disrupted circle or @) an intact circle associated with a disrupted chromosome (which is lethal). The dark arrows represent TnlO elements.

some that haslost the his sequences from ara and retains one copy of TnlO. If intrachromosomal recombination between direct repeats is half-reciprocal, a homologous exchange between the two TnlO elements would generate only one recombinant product: either (1) an intact donor chromosome associated witha disrupted circle (middle of Figure 3) or (2)an intact circle associated with a disrupted donor chromosome, which is presumed to be lethal (bottom of Figure 3). We test the full-recip rocality of recombination by recovering the circular product (see below) and testing the fate of the donor site (at am). Detection of circle formation: Strains in which a circle has formed canbeselected by demanding growth on minimalmedium.Circles generated by either fully reciprocal or half-reciprocal recombination can berecaptured at thehis region of the parental strain which is deleted for theproximal portion of the his operon (Figure 4). A circle to circle recombination event using the hisDC homology of the excised circle,

and thehisDC homology ofthe chromosome will yield a His+ derivative. If a linear fragment were generated from ara, it could not repair the his deletion since the his material present at ara is too short to include homology at theleft side ofthe deleted region (Figure 4). These events are considered in more detail in the APPENDIX.

Use of circle capture to infer reciprocality:Circle integration by recombination requires a fully reciprocal exchange. Therefore the observation ofHis+ recombinants generated by circle integration, in itself, suggests that fully reciprocal recombination can occur. However,sincetheseHis+ recombinants wereselected, their detection gives us no estimate of what fraction of exchanges are fully reciprocal. Wecan assessthis fraction by inspection of the donor site (ma), from which the circle is derived. If intrachromosomal recombination between direct repeats is fully reciprocal, a substantial fraction of the His+recombinants arising by circle integration will be associated with loss of his sequences from the donor ara region and restoration of chromosomalcontinuity (top of Figure 5). If intrachromosomal recombination between direct repeats is half-reciprocal, a homologous exchange between the two TnlO elements will generate only one recombinant product: either (1) an intact chromosome associated with a disrupted circle which cannot be captured; or (with equal probability) (2)an intact circle associated with a disrupted chromosome (bottom of Figure 5). In thelatter case, capture of the circle by recombination with the (broken) donor chromosome would not be detected since we presume this cell would not be viable. Therefore

M. J. Mahan and J. R. Roth

28

Parental Strab (TTI 1791)

1His+

*am

hlrOEOC' 7

w

>

I

D'CEUIIFIE

FIGURE5.-Reciprocal recombination test. Circles generated by fully reciprocal intrachromosomal recombination can integrate into the his region of either: (1) the donor chromosome that generated the circle; or (2) into an uninvolved sister chromosome. Circles generated by half-reciprocal recombination can only be detected if they integrate into a viable uninvolved sister chromosome. If intrachromosomal recombination is fully reciprocal, a substantial fraction of the His+ recombinants would be associated with loss of his sequences at ara. If intrachromosomal recombination is halfreciprocal, all of the His+ recombinants would retain the his sequences at ara.

if recombination between direct repeats is only halfreciprocal, all successful circle capture would be expected to occur by recombination with an uninvolved sister chromosome. Such a recombinant can be identified by possession of his material at the uninvolved ara locus. Results of these tests will be presented below in conjunction with data on inversions. Inversion rearrangements: Since the parental strain (see Figure 2) has his homologiesininverse order,a single fully reciprocal exchange between these homologies should yield a His+ recombinant with an inversion of the chromosomal segment between the his and ara loci (Figure 6). These inversionbearing recombinants can be identified by checking for linkage disruption at the joinpoints of the inversion (Figure 7). Since the inversion contains his material on one side ofeach join point and ara material on the otherside, neither join point can be repaired by a single wild type transduced fragment. However, the transduction frequency inany other region of the chromosome, unlinked to either join point, should be normal. His+ recombinants that show no linkage disruption are classified as examples of circlerecapture. His+ recombinants that show linkage disruption are classifiedasinversions. (Hfr mapping experiments

FIGURE6.-A test for isolating spontaneous inversions of the bacterial chromosome. A portion of the his operon (hisOGDC')is placed at the ara region in the orientation opposite to that of the his operon. The strain contains a his deletion that carries the distal portion of the his operon (hisDChomology is shared by the segment at ara and the mutant his locus). Selection for His+ derivatives yield recombinants some of which have undergone recombination between the separated his homologies. Such recombination results in the inversion of the chromosomal material between the his operon and the site of his homology at ara. The dark arrows represent TnlO elements in the same orientation.

supporting these classifications are presented in the APPENDIX).

Use of inversions to infer reciprocality: The detection of inversions is,in itself, evidence for fully reciprocal recombination betweeninverse repeats. This is true because both ends of the inverted segment mustbe joined to restore chromosomal continuity (Figure 6). If intrachromosomal recombination between inverse order homologies is only half-reciprocal, a homologous exchange between the inverse order repeats would leave a broken chromosome and would be lethal. In such a situation, inversions would not be detected. It should be noted parenthetically that two simultaneous independent half-reciprocalexchangesbetween sister chromosomescan, in principle, also yield

Reciprocality of Genome Rearrangements

111 1077 transduced Iragment

Kmr

X a

X b

his+

,

ec

d

wild type chromosome

U f l

1

Select Km‘

b

/

,

c

d

e

//

Kmr

b

om^^)

Kmr (Ilia’) transductants

TT11077 transduced fragment

noshared

h

l

e

inversion chromosome

1

Select Kmr

Linkage disruption NO Knf tranductants

FIGURE7.-Linkage disruption. P22 phage shown on TTI 1077, which contains a large his deletion associated with a kanamycin resistance determinant (co62l*MudJ*hisF9951),was used to transduce His+ recombinants to kanamycin resistance. Strains containing normal flanking sequences on both sides of the his region will inherit the deletion with normal transduction ability. Strains containing an inverted segment of the chromosome show a reduction in transduction ability at both join points of the inversion (only linkage disruption at the his join point is depicted in the figure). Transduction ability at loci unlinked to either join point is normal. The arrows denote the two breakpoints of the inversion.

an inversion. The low frequency of single sisterstrand exchanges within a 1-2-kb region of homology(- 10-4 estimated from duplication and deletion frequencies) (M. J. MAHANand A. M. SECALL unpublished results), suggests that we would be unable to detect recombinants requiring twosuch independent exchanges (- 10-8). Description of the second parental strain: We have described above one of the parental strains used in this study (TT11791, in Figure 2). This strain (described above) contains the his homology placed at ara in inverse order vis a vis the standard his operon and will be used in studies involving both (1) circle generation and recapture; and (2) inversion rearrangements. A second parent strain ( T T I 1792) is like the first except that the his homology flanked by direct TnlO repeats is inserted atthe pncBlocus (minute 20); the his sequences in the second parent are inserted in the same orientation as the normal his operon. Thissecond strain will be used onlyin studies involving circlecapture, since no inversions canoccur.

29

Both parent strains are phenotypically Hol+His-Tc‘; they can growon histidinol by virtue of the hisOGDC’ chromosomal segment at the ara (or pncB) locus. Fully reciprocal exchanges do occur: Since capture of a circle by recombination requires a fully reciprocal exchange, isolation of His+ derivatives due to circle capture demonstrates that some fully reciprocal exchanges can occur. Similarly, inversions require full reciprocality and their detection demonstrates that fully reciprocal exchanges can occur. Table 2 presents the frequency withwhichHis+derivativesof both typeswereseen for the two parental strains used (TT11791 andT T l l 7 9 2 , described above). We have tested this process of circlegeneration and recapture for avariety of “donor” sites. Circle capture is seen at similar frequency regardless of whether the “donor” his sequences are in direct or inverse order vis a vis the normal his operon (M. J. MAHAN unpublished results). In the case of strain T T l l 7 9 1 (inverse order his material at ma), His+derivativescanalso arise by direct recombination between the his homologies to yield an inversion. The data in Table 2 demonstrate that in this strain, 59% of the His+ derivatives have inversions. In the caseof strain T T l l 7 9 2 with his homology at pncBin direct order vis a vis the his operon, all His+recombinants have occurred by circle capture. In principle, we would have expected that some recombinants might arise by direct recombination of his sequences, generating a duplication. These were not seen. This is not surprising since the predicted duplications would include 75% of the chromosome. Suchduplications are presumably lethaland would certainly be extremely unstable. These results demonstrate that reciprocal exchanges can occur. We have used the circle capture assay to estimate thefraction ofgenetic exchanges that yields both reciprocal products. Full reciprocality is a frequent resultof recombination: By inspecting the fate of the donor site (ara or pncB)in the recombinants that arose by circle capture, we can estimate how frequently generation of a circle is associated with repair of the donor site. A fully reciprocal exchange between the direct repeats generates a circle and repairs the donor site leaving one copy of the direct repeat and no his material. A half-reciprocal exchange that generates a circle would leave an unrepaired (and presumably lethal) chromosome break. In thiscase,His+derivatives will be observed only when the circle integrates into an uninvolved sister chromosome (which hasthe donorsite in its original condition with two direct repeats flanking his material). Recombinants (His+) whichhad trapped a circle at his were examined for the state of their donor site. T o do this, a large deletion was transduced into thehis region, removing all of the his

M. J. Mahan andJ. R. Roth

30

TABLE 2 Frequency of deletion formation, inversion rearrangement, and circle to circle recombination His+ recombinants hisD- segregants per viable cellviable plated"

Parent"TT11791 4.4 hisOGD at ara (minute 2) in inverse orientation to the his o p eron (minute 42). Parent"TT11792 hisOGD at pncB (minute 20) in the same orientation as the his operon (minute 42).

His+ recombinants per cell plated"

10-~

1.2 X 10-4

5.9 X 1 0 - ~

4.3 X 10-5

X

Percent dueto to inversionsb

Percent due have circle captureb

Percent of strains with

captured circles that lost his

sequences from donor site'

(19/53) (53/129) (76/129) 36% 41% 59%

NAd (22/71) 38% (71/71) 100%

* The method for determining the frequency of hisD- segregants and His' recombinants is described in MATERIALS AND METHODS; the frequency was assayed after 24 generations of growth from a single cell. * Inversion rearrangements and circle capture among the His+ recombinants were classified by linkage disruption (see text). ' The Dresence of his seauences at the donor site (ara or was determined as described in MATERIALS AND METHODS. The validity of the methbd is discussed in ;he Appendix. Not applicable.

operon material and thesequences that conferred the His+ phenotype (see MATERIALS AND METHODS). Such transductants can be phenotypically scored for possessionof a functional hisD gene (at ara or pncB) by testing their ability to usehistidinolas a source of histidine. The data in Table 2 show that almost 40% of strains in which a circle was captured (at his) have lost his material from thedonor site. Thus the circle was frequently captured by the same chromosome that generated it and formation of the circle was frequently associated with repair of the donor site. This is expected if recombination is fully reciprocal. The finding that 40% of the His+ clones have lost material from the donor site is consistentwith the idea that the generation of circles is virtually always a fully reciprocal exchange and there is almost a 50% chance of the circle integrating into the same chromosome from whichit emerged. The finding that some of the His+ recombinants retain his material at the donorsite is expected since, evenif the donorsite is always repaired, one would expect that sometimes the freecircle will integrate at the his region of a sister chromosome with a probability dependent on the number of sister nucleoids and theavailability ofthese sites for recombination. The reciprocality, inferred for intrachromosomal recombination events, assesses the ultimate state of the products. That is, the data suggest that both pairs of flanking markers are ultimately joined. However, we cannot infer that both pairsof joins were formed simultaneously by the same event. If, forexample, the initial event joined one pair of flanking sequences, leavingtwo free ends that were later repaired by double strand break repair, this overall process would appear fully reciprocal. This caution, of course, also

applies to all genetic systems in which reciprocality has been inferred. Segregation of a small duplication is frequently due to intrachromosomalrecombinationbetween direct repeats:In theabove sections we have discussed circles formed by recombination between direct repeats on the same chromosome. This leads to loss of the material between these repeats. Loss of such material canalso occur by unequal recombination between sister chromosomes. The observed loss of material between the repeats is therefore due tothe sum oftwo different types of genetic exchange events between regions of direct order homology: (1) intrachromosomal recombination (generating a circle) and (2) interchromosomal recombination generating a deletion in one chromosome and a compensating duplication in the other (Figure 8). When his material between direct repeats is lost without being recap tured elsewhere, loss of the hisD chromosomal segment can be detected as a Hol- segregant, which is still His- but has lost the ability to utilize the intermediate histidinol as a histidine source. These Holsegregants can arise by either circle formation (intrachromosomal) or sister chromosome interaction (interchromosomal). Wehave tried to estimate the relative contributions of these two processes in the following way. Ifit could be shownthat thefrequency of His+ derivatives due to circle capture approached the frequency of Hol- segregants, it would suggest that circles werequantitatively captured, andvirtually every Hol- segregant arises by an intrachromosomal exchange, generating a capturable circle. Initial results (Table 2 andTable 3, line 1) suggested that segregation was much more frequent than circle capture. OneHis+ clone(due tocircle capture) was found per 100 Hol- segregant clones. This ratio could reflect

Reciprocality of Genome Rearrangements

31

tatively recaptured, we would expect to see some Holsegregants due to circle integration after replication of the chromosome and due to integration of the circle into a nonsegregant sister chromosome. Therefore, the numbers observed demonstrate that segregation events generating circles are very frequent. As much as 50% of segregation events could be due to these intrachromosomal events; the rest of the segregation events are presumably due to sister chromosome exchanges.

lnlerchromoromal Recomblnatlon

DISCUSSION

FIGURE8.--Segregation of a duplication. Loss of his material from the uru locus is due to the sum of two different types of recombination events: (1) intrachromosomal recombination between direct repeats resulting in a deletion event (associated with the generation of a free circle); and (2)interchromosomal recombination between direct repeats resulting in a deletion event that is not associated with the generation of a free circle.

inefficient or delayed recapture of circles,or it might suggest that many segregation events occur by exchanges (interchromosomal) that do not generate circles. By increasing the extentof his homology available to permit circle integration, we canshow that the frequency ofHis+ recombinants increases dramatically (Table 3). This was done both by adding his material to the target his operon (line 2), to the pncB donor sites (line3) or by adding material to both (line 4). The frequency of His+ clonesdue tocircle capture increases with the extent of homology, while the frequencyofHolsegregants is unaffected by the changes made. Thus theincrease infrequency of His+ recombinants appears to be due to improved capture efficiency, not to increased generation of circles. With maximumhomology (line 4), we observe one His+ recombinant per 2.5 Hol- segregants. Thus a very high proportion of segregation events generate circles that are subject to recapture. Similar results were obtained by increasing the his homology at the ara locus (data not shown).Even if all segregation occurred by circle generation, and circles were quanti-

The genetic assay described offers several advantages for the analysis of recombination in bacteria. It allows the observation of chromosomalrecombination events in a system where all substrates and recombination functions are exclusively provided by the bacterial chromosome. The assay allows the recovery of both recombinant products from a single recombination event. The assay discerns the full-reciprocality of three different types of recombination events: intrachromosomal recombination between direct and inverse repeats; and circle to circle recombination between an exogenote and thebacterial chromosome (in the absence of any plasmid or phage functions). The test assesses the contribution of fully reciprocalintrachromosomal recombination to the segregation of a small duplication. A summary of our findings is given below. (1) Our results suggestthat alarge fraction of recombinational exchanges are fully reciprocal. (2) Homologous recombination betweeninversehomologiesplaced at variouslocationsof the bacterial chromosome can cause inversion of chromosomal material between the sites of inverted homology. (3) The segregation of a small (10 kb) duplication is frequently dueto an intrachromosomal exchange. One striking aspect ofthe data presented is the high frequency of the “circle capture” recombinant type (5 X 1 O-5). This class requires two exchange events, one within 10 kb of TnlO homology (togeneratethe circle), andone within 1 kbof his homology (to integrate the circle). The frequency ofthisclassis surprising since it is virtually identicalto thefrequency

TABLE 3 Homology dependenceof circle integration

Strain

Genotype

hisD- segregants per viable cell plated

(x 105)

His+ recombinants his homology due to circle ca ture per viable cell pLted‘ available for (x 105) circle cavture ~

TTl1841 TTl1842 TT11843 TTl1844

pncB165::(TnlO-hisOGDC869I-Tn10) hisOGD646 3.2 pncBI65::(TnlO-hisOGDC869I-Tn10) hisOG203 pncB165::(TnIO-hisOGDCBH8698-TnlO) hisOGD646 pncBI65:(TnIO-hisOGDCBH8698-TnIO) hisOG203 240

790 540 660

590

His+ recombinants were scored after 33-35 hr of incubation at 37” on minimal medium.

13 98

hisD’C’ hisC ‘DC ’ hisD ‘CBH’ hisG ‘DCBH’

~~

M. J. Mahan and J. R. Roth

32

TABLE 4 Chromosome transfer of am-his inversion ~~

~~~~~~

~

Gradient of transmission (No. of recombinants)”

Origin of transfer at cob (minute 41)

Origin of transfer

Allele

Recip. TR5686 TR5660 TR5662 TR6976 TR3167 TR5663 TR5661 TR5665 TR5666 TR5667 TR5668 TR5669 TR5670 TR5688 TR567 1 TR5654 TR5655 TR5657 TR5658

aroD140 pyrF146 hisDC2236 hisFI135 metG319 purF145 aroC5 cysC519 serA I3 cysG439 cysE396 ilv-508 metA53 purA155 pyrB64 thrA9 leu-485 purE8 PYrc 7

circle Hol+His-Hol+HisAfter circle inversioncapture parent parent inversioncapture Minuteb TT13793 TT13792 TT13791‘ TT13790 TT13789 TT13788‘ 29 34 42 42 44 49 50 60 63 73 79 83 90 93 98 0 3 12 22

9 33 0 557 1007 567 313 70 265 5 2 5 10 3 16 17 10 1 5

(minute 3)

After

After

After

at l e d

59 84 >3000 >3000 >2000 1603 814 245 629 0 4 45 54 7 180 69 116 4 3

43 76 42 95 36 19 21 10 38

67 988 217 1216 1428 13 2 11

1 1

141 125 0 11 103 22 76 98 154

97 40 42 181 78 36 26 36 121

72 1475 624 >2000 >2000

1344 536 1700

35 20

264 9 2

37 74 852 2064 1104 896 396 194 492 0

16 116 83 71

Hfrs were generated by insertion of F’ 114 ts lac+ zzf-1836::TnlOd-Cam plasmid at a chromosomal MudA(lac, Amp) insertion element in the cob region (first three columns) or l e d gene (last three columns). * To facilitate presentation, recipient markers in Table 4 are arranged vertically in a permutation of the Salmonella chromosome map beginning at minute 29. Donor Hol+ His- parent Hfr strains (TT13788 and TT13791) carry thehisOGD646 deletion at thehis locus and can not repairhisDC2236 to prototrophy because the deletions are overlapping. Arrows indicate the direction of transfer inferred.

of simpleinversion, which requires only one exchange (within 1 kb of his homology). It appears that, once generated, the freecircles havea very high probability of integration (1O-‘). This is incontrast to thegeneral observation that circle-circle recombination is very (LABAN and COHEN inefficient in bacteria ( 1981). We will present evidence suggesting that the high frequency seen is due to activation ofthese circles by a mechanism involving recBC function (M. J. MAHAN and J. R. ROTH manuscript in preparation). This work was supported by U.S. Public Health Service grant GM 27068 from the National Institutes of Health. M. J. M. was supported by predoctoral training grant T32-GM 07464-1 1 from the National lnstitutes of Health.

LITERATURE CITED ANDERSON, P., and J. ROTH, 1981 Spontaneous tandem genetic duplications in Salmonella typhimurium arise by unequal recombination between rRNA (rrn) cistrons. Proc. Natl. Acad. Sci. USA783113-3117. BERKOWITZ, D., J. HUSHON,H. WHITFIELD, J. R. ROTH and B. N. AMES, 1968 Procedure for identifying nonsense mutations. J. Bacteriol. 96: 2 15-220. CAMPBELL, A., 1962 Episomes Adv. Genet. 11: 101-145. CAMPBELL, A., D. BERG,E. LEDERBERG, P. STARLINGER, D.BOTSTEIN,R. NOVICK and W. SZYBALSKI, 1977 Nomenclature of transposable elements on prokaryotes. pp. 15-22. In: DNA

InsertionElements,Plasmids, and Episomes, Edited by I. A. J. A. SHAPIRO and S. L. ADHYA. Cold Spring Harbor BUKHARI, Laboratory, Cold Spring Harbor, N.Y. CHAN, R. K., D. BOTSTEIN,T. WATANABE and Y. OGATA, 1972 Specialized transduction of tetracycline by phage P22 in Salmonellatyphimurium11. Properties of a high frequency transducing lysate. Virology 5 0 883-898. CHUMLEY, F. G., and J. R. ROTH. 1980 Rearrangement of the bacterial chromosome using TnlO as a region of homology. Genetics 9 4 1-14. CHUMLEY, F. G., R. M E N Z E LJ.~ R. ~ ~ROTH, 1979 Hfr formation directed by TnlO. Genetics 91: 639-655. CLARK,A. J., 1973 Recombination deficient mutants of E. coli and other bacteria. Annu. Rev. Genet. 7: 67-86. DAVIS,R. W., D. BOTSTEIN and J. R.ROTH, 1980 Advanced Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. M. E. ADELBERG, A. J. CLARKand P.E. HARTMAN, DEMEREC, 1966 A proposal for a uniform nomenclature in bacterial genetics. Genetics 5 4 61-76. HERMAN, R. K., 1965 Reciprocal recombination of chromosome and F-merogenote in Escherichia coli. J. Bacteriol. 9 0 16641668. HERMAN, R. K., 1968 Identification of recombinant chromosomes and F-merogenotes in merodiploids of Escherichia coli. J. Bacteriol. 9 6 173-179. HUGHES,K. T.,and J. R.ROTH, 1985 Directed formation of deletions and duplications using Mud (Ap, lac). Genetics 109: 263-282. LABAN,A., and A. COHEN,1981 Interplasmidic and intraplas-

Reciprocality of Genome Rearrangements midic recombination in Escherichia coli. Mol. Gen. Genet. 1&4: 200-207. MALOY,S. R., and J. R. ROTH, 1983 Regulation of proline utilization in Salmonella typhimurium:characterization of put::Mud (Ap,lac) operon fusions. J. Bacteriol. 154: 561-568. MFSELSON,M., 1967 Reciprocal recombination in prophage lambda. J. Cell. Physiol. Suppl. 1: 113-1 18. MESELSON,M.,and C. RADDING,1975 A general model for genetic recombination. Proc. Natl. Acad. Sci. USA 74: 358-361. RADDING, C. M., 1982 Homologous pairing and strand exchange in genetic recombination. Annu. Rev. Genet. 1 6 405-437. SANDERSON, K. E., and J. R. ROTH, 1983 Linkage map of Salmonella typhimurium. Edition VI. Microbiol. Rev. 47: 410-452. SARTHY, V., and M. MFSELSON, 1976 Single burst study of Recand Red-mediated recombination in bacteriophage lambda. Proc. Natl. Acad. Sci. USA 73: 46 13-461 7. SCHMID, M. B., and J. R. ROTH,1980 Circularization of transducing fragments: a mechanism for adding segments to the bacterial chromosome. Genetics 94: 15-29. SCHMID, M. B., and J. R. ROTH,1983 Genetic methods for analysis and manipulation of inversion mutations in bacteria. Genetics 105: 517-537. SCHMIEGER, H., 1972 Phage P22- mutants with increased or decreased transduction abilities. Mol. Gen. Genet. 119 75-88. SMITH,G. R., 1983 General Recombination. pp. 175-209. In: J. W. ROBERTS,F. W. Lambda II, Edited byR.W. HENDRIX, STAHLand R. A. WEISBERG. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. STAHL,F. W., 1979a Specialized sites in generalized recombination. Annu. Rev. Genet. 13: 7-24. STAHL,F. W., 1979b GeneticRecombination.ThinkingAbout It in Phage and Fungi. W. H. Freeman, San Francisco. SZOSTAK, J. W., T. L. ORR-WEAVER, R. J. ROTHSTEIN and F. W. STAHL,1983 The double-strand-break repair model for recombination. Cell 33: 25-35. VOGEL,H., and D. BONNER,1956 Acetylornithase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 218: 97-106.

33

FIGURE9.-Duplications formed during generation of His+ recombinants. Recombination between the direct order hisDC homology at pncB and the hisDC homology at the his locus generates a duplication having his+ sequences at the join point. The dark lines underscore the homology available for therecombination event.

since the circle wouldinclude a single copy of the TnlO element. One might expect that the small duplication generated by circle integration would be unstable and give rise to His-, Tets segregant clones. This is difficult to test because duplications with such short repeats as these (about 1 kb) segregate rarely. Communicating editor: E. W. JONFS We have detected such segregants but they are found at a frequency of only -1110~ cells. APPENDIX Failure to detect large duplications: In the case of strains with direct order repeats of his homology, we expected to Evidence for capture of a circleat the his locus: Capture of recover His+ recombinants carrying the large duplication exa circle a t his by recombination within hisDC homology predicts tending fromhis t o pncB (across the origin ofreplication); such the generation of a short duplication including the hisD gene. recombinants would carry their his+ material at the join point Severallines of evidence make us confident that thiscircle (Figure 9). T h e recombinants would be expected to be highly (hisOG203 or capturehasoccurred.Thedeletionmutation hisOGD646) present a t t h ehis locus inthe parent strains extends unstable and possibly deleterious. We have sought such duplications among our His+ recombinants using methods developed to the left (Figure 4) beyond the length of his homology placed and ROTH (1 98 1). Ten independentHis+ recomby ANDERSON a t uru or pncB. T h u s a linear fragment derived from the donor binants were tested; none carries the predicted large duplicasite cannot generate a His+ recombinant because it only has tion. Furthermore among the many His+recombinants studied, homology with the chromosome at the right side of the deletion. we have not observed an unstable recombinant type that might A circular fragment can be added to thehis locus by recombibe due tosuch duplications. Finally, the tests done toassess the nation with homology on only one side of the recipient deletion state of the pncB locus could have revealed thelarge duplication (see Figure 4). type as His+ clones which do notbecome His- after introduction We have scored recombinants for possession of inversions of the large his deletion used in this test. No such strains were and duplications(see below). T h e test for these rearrangements observed. Therefore while large duplications are a formally detects linkage disruption at the his locus. T h e recombinants possible way ofgenerating a His+ derivativefromstrain we have attributed to circle recapture carryhis+ material at the T T I 1792, we find no evidence that such events have occurred. normal his locus and show n o linkage disruption there, asjudged It is possible that strains with this duplication are inviable or by the fact that the large his*MudJ*cob deletion can be introare so unstable that they lose their his+ material (which would duced with high efficiency, replacing the his+ material. This be present at the duplication join point) tooquickly to survive suggests that material essential to the His+ phenotype has been his locus with normal flanking sequences on both even under selective conditions. inserted at the sides. Other transductioncrosses have demonstrated that "circle Alternative chromosomal rearrangements: We have concapture" recombinants have acquired a Tc' determinant at the cluded that someHis+ recombinants have arisen by excision of his locus; this is predicted if the circle integration has occurred, a small circle of material from the ara site and integration of

34

M. J. Mahan and J. R. Roth

that circle at his. It is formally possible that recombinants of the same final structure could have arisen by two successive rearrangements generated by direct interaction of chromosomal sequences. In the case of strains with his sequences in inverse order, this could occur by an initial inversion event between his sequences, followed by a second inversion event between TnlO elements that restores the normal chromosome order and leaves a his+ operon at thehis locus. The recombination events required for successive inversions are the same as those required for circle generation and recapture, one between TnlO elements (10 kb) and onebetween the shared his homology (1 kb). Three lines of evidence lead us to conclude that this recombinant class arises predominately by circle formation and c a p ture. 1. If recombinants of this type arose only by sequential inversions, they would all lack his sequences at ara. In fact, more than half of these recombinants retain his material at ara and must have arisen in a different way. 2. The frequency of the “circle capture” recombinants is the same regardless of the orientation of the his cassette (TnlOhisOGDC-TnlO). This is expected if the recombinants arise by circle formation and capture. This is not expected if this class arises by sequential rearrangements. In the case ofhis sequences in inverse order (at a m ) , the intermediate is an inversion, which is viable, and we have shown to have a nearly normal growth rate; in the case of his sequences in direct order (at pncB), the duplication intermediate is one we have never observed (see above) and is either inviable or very short-lived. Thus, with sequential rearrangement, we would expect parent strains to differ widely in the frequency of the “circle capture” class. In fact, we observe that the frequency of the “circle capture” class is similar for parent strains with his sequences in inverse or direct order; we have tested this for a variety of chromosomal sites in addition to the sites presented here (M. J. MAHAN unpublished results). 3. The relative frequency of His+ recombinant types (circle capture compared to inversions) remains constant, while the overall frequency of His+ recombinants increases during the growth of the parental strain (data not shown). If sequential inversions were needed to generate the “capture” class,we would expect the inversion class to be higher initially and the capture class to increase in relative frequency with time. Even if the class wehave attributed to circle captured proved to be due, completely or in part, to sequential inversions, the overall conclusions drawn would not be affected. The bulk of the chromosomal recombination events scored are fully reciprocal. Test of the state of the donor site does reflect material at ara: In Table 2 we concluded that asubstantial number of His+ recombinants have lost his sequences from their ara region. Several genetic tests haveconfirmed our interpretation of these tests. T o reveal the genotype of the ara locus, P22 grown on his deletion strain TT11077 (cob2l*MudJ*hisF9951) was used to transduce six independent His+ recombinants derived from parent strain T T l l 7 9 1 (ara::(TnlO-hisOGDC-TnlO)),to kanamycin resistance. This selection requires inheritance of a large deletion replacing the recipient his region. These Km’ transductants were of two phenotypic classes: Hol+His-Tc‘ (class 1) and Hol-HisTc’ (class 2). We have inferred from such results that class 1 strains have hisD+ material at ara and class 2 strains d o not. T o test this interpretation, both classes of Km‘ transductants were transduced to Ara+ with P22 phage grown on LT2 to check whether the hisD material (conferring the Hol+ phenotype) is, infact, at ara. All Ara+ transductants (200/200) isolated from fourindependent class 1 (Hol+His-Tc‘Km‘) recipients

exhibited a Hol-His-Tc‘Km’ phenotype, demonstrating that both a TnlO element and a functional hisD gene were present at the ara locus.Similarly,all Ara+ transductants (200/200) isolated from five independent class 2 (Hol-His-Tc‘Km’) recipients also exhibited a Hol-His-Tc‘Km’ phenotype. Thus, class 2 Km’ transductants harbor only a TnlO insertion at ara as expected for strains that had lost his sequences by recombination. Parallel experiments were performed to reveal the genotype of the pncB donor region following circle capture. P22 phage grown on his deletion strain TT11077 (cob2l*MudJ*hisF9951) was used to transduce nine independent His+ recombinants derived from strain TT11792 (pncB::(TnlO-hisOGDC)-TnlO)to kanamycin resistance. Again, class 1 and class 2 Km’ transductants were isolated (see above). P22 phage grown on TT8057 (pncB22O::MudA) was used to transduce both classes to ampicillin resistance. As was demonstrated above, the inheritance of the insertion element by Class 1 Km’ transductants was associated with loss of both the Hol+ and Tc’ phenotypes; the inheritance of the insertion element by class 2 Km‘ recipients was associated with loss of the Tc‘ phenotype. Loss of sequences from ara is not due to a second indeWe have concluded that circle pendentsegregationevent: generation and recapture is frequently associated with loss of his sequences from ara (or pncB). This conclusion would be in error if the loss of his material from ara occurred by a second independent recombination event (between the flanking direct repeats at ara) stimulated by the transduction event involved in the assay procedure. The primary argument against an independent event is that spontaneous loss of his material at ara occurs rarely (4.4 X lo-’; see Table 2) while roughly 50% of the His+ recombinants are found to lack his material at ara. It might be argued that all His+ recombinants initially have his material at a m , but the transduction event used to assay the presence of his material at ara stimulates secondary segregation. In order to address this objection, we tested the effect of distant recombination events on the frequency of segregation events at a m . P22 grown on deletion strain TT11077 (cob2l*MudJ*hisF9951) was used to transduce four independent His+ recombinants having hisD+ sequences at ara. All Km’ transductants (200 of 200) derived from each offour independent His+ recombinant recipients retained the hisD sequences at a m . Thus segregation is not markedly stimulated. Similar experiments were performed on five independent His+ recombinants that retained the hisD+sequences at pncB. Identical results were obtained; all Km’ transductants (200 of 200) derived from each of the five independent His+ recombinant recipients retained the hisD sequences at arcs. These results suggest that the proposed stimulation does not contribute significantly to the loss of hisD sequences scored in the reciprocal recombination assay. Hfr mapping of thechromosomalinversion: The His+ recombinants that showed linkage disruption at his and ara were inferred to carry an inversion of the chromosomal segment between the ara and his regions. This conclusion was confirmed by Hfr mapping. Hfr strains were selected from parental strains containing an F‘114 ts lac+ zzf-1836::TnlOd-Cam plasmid and a chromosomal MudA(lac, Amp)insertion element in either the cob region (minute 41) or the leuA gene (minute 3). Thermoresistant, chloramphenicol resistant Hfr derivatives were selected by homologous recombination between the lac homology in the episome and lac homology inthe chromosome [provided by the MudA(lac, Amp) insertion element (CHUMLEY,MENZEL and ROTH 1979, MALOYand ROTH 1983)l. The Hfr strains were used as donors in conjugation crosses with several auxotrophic recipients. Prototrophic recombinants for each of the recipient markers were scored. Table 4 presents the gradient of transmission of chromosomal markers from

Reciprocality 35Rearrangements of Genome donor Hfr strains in the cob region (first three columns) or the leuA gene (last three columns). The Hfrstrains were generated in three geneticbackgrounds: (1) Hol+His- parent; (2) His+ recombinant after circle capture (normal chromosome structure); and (3) His+ recombinant after ara-his inversion. Inspection of thedata in Table 4 reveals that inversion strain TT 13790, with an origin of transfer in the cob region at minute

41, initiates chromosomal transfer counterclockwise, starting with the thrA marker at minute 0. Inversion strain TT13793, with an origin of transfer in the leuA gene at minute 3, initiates chromosomal transfer clockwise, starting with the hisDC marker at minute42. These data confirm the ara-his inversion structure predicted by the linkage disruption test described earlier in RESULTS.