reciprocality of recombination events that rearrange the ...infection or the presence of a foreign...

Copyright 0 1988 by the Genetics Society of America

Reciprocality of Recombination Events That Rearrange the Chromosome

Michael J. Mahan and John R. 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 exclusively by the bacterial chromosome. The genetic system allows the recovery of both recombinant products from 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 suggest that intrachromosomal recombination in bacteria is frequently fully reciprocal.

I 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) (CLARK 1973; MESELSON and RADDING 1975; STAHL 1979a,b; RADDING 1982; SMITH 1983; 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 generated by 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 one circle 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 of recombination in bacteria. A statistical analysis was performed on the recombinant progeny derived from genetic crosses involving multiply marked lambda chromosomes present either in double lysogens (MESELSON 1967), or in lambda lytic crosses (SARTHY and MESELSON 1976). The results from both types of crosses yielded reciprocal recom-

Genetics 120: 23-35 (September, 1988)

bination types at equal frequencies even in single bursts, leading to the conclusion that under 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 distin- guish between fully reciprocal 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 cross- over resulting in the formation of an Hfr strain with a duplication of the sequences at the site of integration. The reversal of this process regenerates the original F’ episome (CAMPBELL 1962). The results of these types of 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 the events observed.

Thus, there are inherent complications in testing reciprocality of bacterial recombination. (1) The products of one event are not usually held together. (2) Most test systems perturb the cell in some way, by subjecting it to infection, conjugation, or transduction and by introducing foreign functions that may contribute to the course of the event. (3) Many test systems present substrates that may not normally be seen in a cell such as double strand ends, atypical supercoiling, or single stranded substrates.

24 M. J . Mahan and J . R. Roth

r u l l Y - g e r t r r c n r p 1 A B

X a b

\ a b

A B

/ a b

A b

_I__) a B

A b - __I)

a B

FIGURE 1 .-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 some of these problems through use of a genetic system in which all substrates 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 inter- and 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 (CHUMLEY and ROTH 1980) and (SCHMID and 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 (HUGHES and 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 the join 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 auxo- trophs, 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 (TT 1 183 1) and used to transduce LT2 to tetracycline resistance. Five Tc' His+ transductants were isolated. The prototrophic transductants were grown overnight in liquid nutrient broth medium nonselectively in order to allow loss of the wild type his copy, leaving a segregant clone with a haploid copy of hisOGD646 that had been introduced into an LT2 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 at 37'. 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 cell generations was calculated (N = N0F, where x is the number of cell generations, NO is 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). Pop- ulations tested further (see below) had undergone 24 * 1 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 printed to (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 colonies continue to appear with extended incubation times, 38 hr of incubation at 37' was arbitrarily chosen for assaying colony number. The number of colonies arising on the plate was found to be dependent on the extent of residual growth on the selection medium and thus on the residual nutrients plated. To make this 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 throughout the 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 HUGHES and 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 the distant 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

Reciprocality of Genome Rearrangements 25

TABLE 1

Bacterial strains

Strain Genotype Method“ Recipient Donor

TR6535 TR6976 T T 5 13 TT1151 TT1704 TT3003 T T 3 164 TT3291 TT3699 TT62 12 TT7701 TT8057 TT10286 TTllO77 TT11782 T T l l 7 8 3 T T l l 7 8 5 T T l l 7 8 6 T T l l 7 8 7 T T l l 7 8 8 TT11789 T T l l 7 9 0 T T l l 7 9 1 T T l l 7 9 2 T T l l 7 9 3

T T l l 7 9 7

TT11830 TT11831

T T l l 8 3 2

T T l l 8 3 3

TT 1 1834 T T l l 8 3 5

T T l l 8 3 6 T T l l 8 4 1 TT11842 TT11843 T T l l 8 4 4 TT12914 TT13788

TT13789

TT13790

TT13791

TT13792

TT13793

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)]

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[(hisOGD646-

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

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,

ara-65l::(TnlO-hisO-C869l-TnlO), DUP764[(hisOGD646-

ly~J8l::MudF

C8691)*TnlO*(hisO-E+)], proAB47

OGD646)], zee-2:TnlO,proAB47

F‘114 ts lac+ zrf-1836::TnlOd-Cam

C8691)*TnlO*(hisO-E+)], proAB47, cob-3O::MudA, F’114 ts lac+ zrf-1836::TnlOd-Cam

OGD646)], proAB47,cob-30::MudA, zee-P::TnlO, F’114 ts lac’ zrf-1836::TnlOd-Cam

ara-651::(TnlO-hisO-C8691-TnlO), proAB47, leu- All79::MudA, F’114 ts lac+zzf1836::TnlOd-Cam

ara-651::(TnlO-hisO-C8691-TnlO), DUP764[(hisOGD646- C8691)*TnlO*(hisO-E+)], proAB47, leuAl179::MudA, F’114 ts lac+ zrf-1836::TnlOd-Cam

OGD646)], tee-a::TnlO, leuAl179:MudA, proAB47, zee- 2:TnlO, F’114 ts lac+ zrf-1836::TnlOd-Cam

INV768[(ara-651::TnlO)*his+*(hisC8691::TnlO-his-

INV768[(ara-651::TnlO)*his+*(hisC8691::TnlO-his-

T hisOGD646 proAB47 T his135 TR5667 T hisOG203 Pooled TnlO insertion T 1t2 Pooled TnlO insertion TnlO generated HisG- from zee-1::TnlO T hisOGD646 TT 1 15 1 T hisC129 TT5 13 (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 TT10377 TT12116 T TT3003 TT3 164 T TT 1704 TT3699 T TTl704 TT62 12 T TT11785 TT11783 T TT11786 TT11783 T TTll785 TT3291 T TT11786 TT3291 T TR6535 T T l l 7 8 7 T TR6535 TT 1 1788 His+ revertant of TT 1 I79 1

His+ revertant of TT 1 179 1

T 1t2 T T l l 8 5 3 T hisOGD646 T T l l 8 3 0

T hisOG203 T T l l 8 3 0

T 1t2 TT11831

Tc” His- segregant of T T l l 8 3 3 T 1t2 T T l l 8 2

Tc’ His- segregant of T T l l 8 3 5 T TT11834 TT11788 T T T l l 8 3 6 T T l l 7 8 8 T T T l l 8 3 4 T T l l 7 9 0 T TT11790 TT11790 T TR2647 TT10604 C TT13652 TT12914

C TT13653 TT12914

C TT13654 TT12914

C TT13655 TT12914

C TT13656 TT12914

C TT13657 TT12914

~

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.

26 M. J. Mahan and J. R. Roth

by scoring the ability of the Km’ transductants (which must inherit the deletion) to utilize histidinol (Hol) as a histidine 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 in DEMEREC et al. (1966), CAMPBELL et al. (1977), and CHUMLEY, MENZEL and ROTH (19’79). The nomenclature z--::TnlO refers to a TnlO 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 (1 983), and more recently in HUGHES and ROTH (1 985).

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 BER- KOWITZ et al. (1968), supplemented with 0.2% of the appro- priate carbon source. The complex medium was nutrient broth (8 g/liter, Difco Laboratories) with added NaCl (5 g/ liter). Solid medium contained Difco agar at 1.5% final concentration. Auxotrophic requirements were included in media at final concentrations described by DAVIS, BOTSTEIN, and ROTH (1980). Final concentrations of antibiotics were: tetracycline hydrochloride (Sigma Chemical Co., 16 pg/ml in rich medium, or 10 pg/ml in minimal medium); kanamycin sulfate (Sigma Chemical Co., 50 pg/ml in rich medium, or 100 pg/ml in minimal medium); ampicillin (Sigma Chemical Co., 40 pg/ml in rich medium, or 15 pg/ml in minimal medium), chloramphenicol (Sigma Chemical Co., 20 pg/ml in rich medium or 5 pg/ml in minimal medium); 6-amino-nicotinic acid (Sigma Chemical Co., 50 pg/ml minimal medium).

Transductional methods: The high frequency generalized transducing bacteriophage P22 mutant H T 105/1, int- 201 (SCHMIEGER 1972) was used for all transductional crosses. Unless otherwise specified, 0.1 ml of an overnight culture grown in complex media (ca. 2-4 X lo9 colony- forming units/ml) was used as a recipient of 0.1 ml transducing phage (ca. 108-109 plaque-forming units/ml) and plated directly on selective plates. Transductional crosses involving the selection of kanamycin or chloramphenicol resistance were preincubated overnight on solid nonselec- tive complex medium, then replica printed onto selective medium. Transductants were purified and phage-free iso- lates were obtained by streaking for single colonies on green indicator plates (CHAN et al. 1972). Phage-free colonies were tested for phage sensitivity by cross streaking with P22 H5 (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

Parental Strain (TT11791)

FIGURE 2.-The parental strain used in the reciprocal recombination assay. The ara region (TTI 179 1 ) contains a hisOGDC’ chromosomal segment flanked by two TnfO elements (represented by solid arrows) in the same orientation (C’ indicates that only part of the hisC gene is present here). The parent strain also contains a deletion in its standard his region (hisOGD646). The his sequences are placed at ara in inverse order with respect to the orientation of the normal his operon.

One of the parent strains used in this test (TT11791) is diagrammed in Figure 2. The ara region contains the proximal portion of the his operon flanked by two TnlO elements in the same orientation; the his material present at ara is in inverse orientation vis (I vis the standard his operon. The presence of his material at ara may be scored genetically due to the presence of a functional hisD gene. The parent strain carries a deletion in its standard his region that removes the his promoter and the proximal portion of the his operon, including part of the hisD gene. The phenotype of the parent strain is His- since it lacks an expressed hisC gene. The strain is able to grow on histidinol as a histidine source (Hal+) due to expres- sion of the hisD gene in the chromosomal segment located at ara. Events that can be detected in this strain are described below.

Deletion formation and circle generation: The test for the reciprocality of recombination between direct repeats involves the loss of his sequences from ara, generating a circle, and the recapture of 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-


broken clrcle + - intact chromosome

(Case 2)

intact clrcle + - + broken chromosome

FIGURE 3.--Generation of free excised circles. Circles may be generated by either fully reciprocal or half-reciprocal recombination. Fully reciprocal intrachromosomal recombination between direct repeats at 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 donor site. Half- reciprocal 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 has lost 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 with a 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 can be selected by demanding growth on minimal medium. Circles generated by either fully reciprocal or half-reciprocal recombination can be recaptured at the his region of the parental strain which is deleted for the proximal portion of the his operon (Figure 4). A circle to circle recombination event using the hisDC homology of the excised circle,

No hom0lOgy with clrcle

captured ctrcle a t RfS

FIGURE 4.-Recapture of the circular product. Circles generated by either fully reciprocal or half-reciprocal intrachromosomal recombination may be recaptured at the his operon. A circle to circle recombination event involving the hisDC homology of the free circle and the hisDC 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.

and the hisDC homology of the 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 the left side of the deleted region (Figure 4). These events are considered in more detail in the

Use of circle capture to infer reciprocality: Circle integration by recombination requires a fully reciprocal exchange. Therefore the observation of His+ recombinants generated by circle integration, in itself, suggests that fully reciprocal recombination can occur. However, since these His+ recombinants were selected, their detection gives us no estimate of what fraction of exchanges are fully reciprocal. We can assess this 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 chromosomal continuity (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 the latter 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

APPENDIX.


hlrOEOC' *am > I D'CEUIIFIE 7 w

FIGURE 5.-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 half- reciprocal, all of the His+ recombinants would retain the his sequences at ara.

P a r e n t a l S t r a b (TTI 1791)

1 His+

if recombination between direct repeats is only half- reciprocal, 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 homologies in inverse 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 inversion- bearing recombinants can be identified by checking for linkage disruption at the join points of the inversion (Figure 7). Since the inversion contains his material on one side of each join point and ara material on the other side, neither join point can be repaired by a single wild type transduced fragment. However, the transduction frequency in any 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 circle recapture. His+ recombinants that show linkage disruption are classified as inversions. (Hfr mapping experiments

FIGURE 6.-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 (hisDC homology 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 between inverse repeats. This is true because both ends of the inverted segment must be 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 simul- taneous independent half-reciprocal exchanges between sister chromosomes can, in principle, also yield


Kmr 111 1077 transduced Iragment

X X a his+ b

U f l

1 , c d e wild type

chromosome

Select Km‘

b / , c d e / /

Kmr (Ilia’) transductants

Kmr b

TT11077 transduced fragment

no shared

h l om^^) e

chromosome inversion

1 Select Kmr

Linkage disruption NO Knf tranductants

FIGURE 7.-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 sister strand exchanges within a 1-2-kb region of homology (- 1 0-4 estimated from duplication and deletion frequencies) (M. J. MAHAN and A. M. SECALL unpublished results), suggests that we would be unable to detect recombinants requiring two such independent exchanges

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 (TTI 1792) is like the first except that the his homology flanked by direct TnlO repeats is inserted at the pncB locus (minute 20); the his sequences in the second parent are inserted in the same orientation as the normal his operon. This second strain will be used only in studies involving circle capture, since no inversions can occur.

(- 10-8).

Both parent strains are phenotypically Hol+His-Tc‘; they can grow on 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 with which His+ derivatives of both types were seen for the two parental strains used (TT11791 and TTll792, described above). We have tested this process of circle generation and recapture for a variety 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 TTl l791 (inverse order his material at ma), His+ derivatives can also 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 case of strain TTl l792 with his homology at pncB in 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. Such duplications are presumably lethal and would certainly be extremely unstable.

These results demonstrate that reciprocal exchanges can occur. We have used the circle capture assay to estimate thefraction of genetic exchanges that yields both reciprocal products.

Full reciprocality is a frequent result of 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 this case, His+ derivatives will be observed only when the circle integrates into an uninvolved sister chromosome (which has the donor site in its original condition with two direct repeats flanking his material). Recombinants (His+) which had trapped a circle at his were examined for the state of their donor site. To do this, a large deletion was transduced into the his region, removing all of the his


TABLE 2

Frequency of deletion formation, inversion rearrangement, and circle to circle recombination

His+ recombinants Percent of strains with captured circles that

Percent due have lost his

viable cell plated" viable cell plated" inversionsb captureb donor site' sequences from hisD- segregants per His+ recombinants per Percent due to to circle

Parent"TT11791 4.4 X 1 0 - ~ 1.2 X 10-4 59% (76/129) 41% (53/129) 36% (19/53) hisOGD at ara (minute 2) in inverse orientation to the his o p eron (minute 42).

hisOGD at pncB (minute 20) in the same orientation as the his operon (minute 42).

* The method for determining the frequency of hisD- segregants and His' recombinants is described in MATERIALS AND METHODS; the

* 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

Parent"TT11792 5.9 X 1 0 - ~ 4.3 X 1 0 - 5 NAd 100% (71/71) 38% (22/71)

frequency was assayed after 24 generations of growth from a single cell.

the methbd is discussed in ;he Appendix. Not applicable.

operon material and the sequences that conferred the His+ phenotype (see MATERIALS AND METHODS). Such transductants can be phenotypically scored for possession of a functional hisD gene (at ara or pncB) by testing their ability to use histidinol as 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 the donor 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 consistent with 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 which it emerged. The finding that some of the His+ recombinants retain his material at the donor site is expected since, even if the donor site is always repaired, one would expect that sometimes the free circle will integrate at the his region of a sister chromosome with a probability dependent on the number of sister nucleoids and the availability of these 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 pairs of joins were formed simultaneously by the same event. If, for example, the initial event joined one pair of flanking sequences, leaving two 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 intrachromosomal recombination between direct repeats: In the above 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 can also occur by unequal recombination between sister chromosomes. The observed loss of material between the repeats is therefore due to the sum of two 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 Hol- segregants can arise by either circle formation (intrachromosomal) or sister chromosome interaction (interchromosomal). We have tried to estimate the relative contributions of these two processes in the following way. If it could be shown that the frequency of His+ derivatives due to circle capture approached the frequency of Hol- segregants, it would suggest that circles were quantitatively captured, and virtually every Hol- segregant arises by an intrachromosomal exchange, generating a capturable circle. Initial results (Table 2 and Table 3, line 1) suggested that segregation was much more frequent than circle capture. One His+ clone (due to circle capture) was found per 100 Hol- segregant clones. This ratio could reflect


lnlerchromoromal Recomblnatlon

FIGURE 8.--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 extent of his homology available to permit circle integration, we can show that the frequency of His+ recombinants increases dramati- cally (Table 3). This was done both by adding his material to the target his operon (line 2), to the pncB donor sites (line 3) or by adding material to both (line 4). The frequency of His+ clones due to circle capture increases with the extent of homology, while the frequency of Hol- segregants is unaffected by the changes made. Thus the increase in frequency of His+ recombinants appears to be due to improved capture efficiency, not to increased generation of circles. With maximum homology (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-

tatively recaptured, we would expect to see some Hol- segregants due to circle integration after replication of the chromosome and due to integration of the circle into a nonsegregant sister chromosome. There- fore, 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.

DISCUSSION

The genetic assay described offers several advan- tages for the analysis of recombination in bacteria. It allows the observation of chromosomal recombination 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 the bacterial chromosome (in the absence of any plasmid or phage functions). The test assesses the contribution of fully reciprocal intrachromosomal recombination to the segregation of a small duplication.

A summary of our findings is given below. (1 ) Our results suggest that a large fraction of recombinational exchanges are fully reciprocal. (2) Homologous recombination between inverse homologies placed at various locations of 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 due to an intrachromosomal exchange.

One striking aspect of the 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 (to generate the circle), and one within 1 kb of his homology (to integrate the circle). The frequency of this class is surprising since it is virtually identical to the frequency

TABLE 3

Homology dependence of circle integration

hisD- segregants due to circle ca ture his homology

Strain Genotype (x 105) (x 105) circle cavture per viable cell plated per viable cell pLted‘ available for

His+ recombinants

~

TTl1841 pncB165::(TnlO-hisOGDC869I-Tn10) hisOGD646 790 3.2 hisD’C’ TTl1842 pncBI65::(TnlO-hisOGDC869I-Tn10) hisOG203 540 13 hisC ‘DC ’ TT11843 pncB165::(TnIO-hisOGDCBH8698-TnlO) hisOGD646 660 98 hisD ‘CBH’ TTl1844 pncBI65:(TnIO-hisOGDCBH8698-TnIO) hisOG203 590 240 hisG ‘DCBH’

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

~~


TABLE 4

Chromosome transfer of am-his inversion ~~

Gradient of transmission (No. of recombinants)”

Origin of transfer at cob (minute 41) Origin of transfer at l e d (minute 3)

~~~~~~ ~

Hol+His- After circle After Hol+His- circle After

parent Recip. Allele

capture inversion parent capture inversion Minuteb TT13788‘ TT13789 TT13790 TT13791‘ TT13792 TT13793

After

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 cysC5 19 serA I3

cysE396 ilv-508 metA53 purA155 pyrB64 thrA9 leu-485 purE8

cysG439

PYrc 7

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

59 84

>3000 >3000 >2000

1603 814 245 629

0 4

45 54 7

180 69

116 4 3

43 141 97 37 76 125 40 74 42 0 42 852 95 11 181 2064 36 103 78 1104 19 22 36 896 21 76 26 396 10 98 36 194 38 154 121 492

0

67 988 1475 1344 72 217 624 536 16

1216 >2000 1700 116 1428 >2000 83

13 264 71 2 35 9 1

11 20 2 1

Hfrs were generated by insertion of F’ 114 ts lac+ zzf-1836::TnlOd-Cam plasmid at a chromosomal MudA(lac, Amp) insertion element

* To facilitate presentation, recipient markers in Table 4 are arranged vertically in a permutation of the Salmonella chromosome map

Donor Hol+ His- parent Hfr strains (TT13788 and TT13791) carry the hisOGD646 deletion at the his locus and can not repair hisDC2236

in the cob region (first three columns) or l e d gene (last three columns).

beginning at minute 29.

to prototrophy because the deletions are overlapping. Arrows indicate the direction of transfer inferred.

of simple inversion, which requires only one exchange (within 1 kb of his homology). It appears that, once generated, the free circles have a very high probability of integration (1 O-‘). This is in contrast to the general observation that circle-circle recombination is very inefficient in bacteria ( (LABAN and COHEN 1981). We will present evidence suggesting that the high frequency seen is due to activation of these circles by a mechanism involving recBC function (M. J. MA- HAN 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

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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.

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CLARK, A. J., 1973 Recombination deficient mutants of E. coli and other bacteria. Annu. Rev. Genet. 7: 67-86.

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DEMEREC, M. E. ADELBERG, A. J. CLARK and P. E. HARTMAN, 1966 A proposal for a uniform nomenclature in bacterial genetics. Genetics 5 4 61-76.

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Communicating editor: E. W. JONFS

APPENDIX

Evidence for capture of a circle at the his locus: Capture of a circle a t his by recombination within hisDC homology predicts the generation of a short duplication including the hisD gene. Several lines of evidence make us confident that this circle capture has occurred. The deletion mutation (hisOG203 or hisOGD646) present a t the his locus in the parent strains extends to the left (Figure 4) beyond the length of his homology placed at uru or pncB. Thus a linear fragment derived from the donor site cannot generate a His+ recombinant because it only has homology with the chromosome at the right side of the deletion. A circular fragment can be added to the his locus by recombination with homology on only one side of the recipient deletion (see Figure 4).

We have scored recombinants for possession of inversions and duplications (see below). T h e test for these rearrangements detects linkage disruption at the his locus. T h e recombinants we have attributed to circle recapture carry his+ material at the normal his locus and show no linkage disruption there, asjudged by the fact that the large his*MudJ*cob deletion can be introduced with high efficiency, replacing the his+ material. This suggests that material essential to the His+ phenotype has been inserted at the his locus with normal flanking sequences on both sides. Other transduction crosses have demonstrated that "circle capture" recombinants have acquired a Tc' determinant at the his locus; this is predicted if the circle integration has occurred,

FIGURE 9.-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 the recombination event.

since the circle would include 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. We have detected such segregants but they are found at a frequency of only -1110~ cells.

Failure to detect large duplications: In the case of strains with direct order repeats of his homology, we expected to recover His+ recombinants carrying the large duplication ex- tending from his to pncB (across the origin of replication); such recombinants would carry their his+ material at the join point (Figure 9). The recombinants would be expected to be highly unstable and possibly deleterious. We have sought such duplications among our His+ recombinants using methods developed by ANDERSON and ROTH ( 1 98 1). Ten independent His+ recombinants were tested; none carries the predicted large duplication. Furthermore among the many His+ recombinants studied, we have not observed an unstable recombinant type that might be due to such duplications. Finally, the tests done to assess the state of the pncB locus could have revealed the large duplication type as His+ clones which do not become His- after introduction of the large his deletion used in this test. No such strains were observed. Therefore while large duplications are a formally possible way of generating a His+ derivative from strain T T I 1792, we find no evidence that such events have occurred. It is possible that strains with this duplication are inviable or are so unstable that they lose their his+ material (which would be present at the duplication join point) too quickly to survive even under selective conditions.

Alternative chromosomal rearrangements: We have concluded that some His+ recombinants have arisen by excision of a small circle of material from the ara site and integration of


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 the his locus. The recombination events required for successive inversions are the same as those required for circle generation and recapture, one between TnlO elements (1 0 kb) and one between 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 (TnlO- hisOGDC-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 of his sequences in inverse order (at am), 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 we have 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 a substantial number of His+ recombinants have lost his sequences from their ara region. Several genetic tests have confirmed 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 TTl l791 (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 do 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, in fact, at ara. All Ara+ transductants (200/200) isolated from four independent 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 independent segregation event: We have concluded that circle 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 am. 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 of four independent His+ recombinant recipients retained the hisD sequences at am. 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 the chromosomal inversion: 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). Thermo- resistant, chloramphenicol resistant Hfr derivatives were selected by homologous recombination between the lac homology in the episome and lac homology in the chromosome [provided by the MudA(lac, Amp) insertion element (CHUMLEY, MENZEL and ROTH 1979, MALOY and 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


donor Hfr strains in the cob region (first three columns) or the 41, initiates chromosomal transfer counterclockwise, starting leuA gene (last three columns). The Hfr strains were generated with the thrA marker at minute 0. Inversion strain TT13793, in three genetic backgrounds: (1) Hol+His- parent; (2) His+ with an origin of transfer in the leuA gene at minute 3, initiates recombinant after circle capture (normal chromosome struc- chromosomal transfer clockwise, starting with the hisDC marker ture); and (3) His+ recombinant after ara-his inversion. Inspec- at minute 42. These data confirm the ara-his inversion structure tion of the data in Table 4 reveals that inversion strain predicted by the linkage disruption test described earlier in TT 13790, with an origin of transfer in the cob region at minute RESULTS.

reciprocality of recombination events that rearrange the ...infection or the presence of a foreign...

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