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Approaches To-Half-Tetrad Analysis in Bacteria

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Approaches To-Half-Tetrad Analysis in Bacteria Copyright 0 1994 by the Genetics Society of America Approaches to-Half-TetradAnalysis in Bacteria: Recombination Between Repeated, Inverseorder Chromosomal Sequences Anca M. %gall’ and JohnR. Roth Department of Biology, University of Utah, Salt Lake City, Utah 841 12 Manuscript received March 26, 1993 Accepted for publication October 5, 1993 ABSTRACT In standard bacterial recombination assays, a linear fragment of DNA is transferred to a recipient cell and, at most, a single selected recombinant typeis recovered from each merozygote. This contrasts with fungal systems, for which tetrads allow recovery of all meiotic products, including both ultimate recombinant products of an apparent single act of recombination. We have developed a bacterial recombination system in which two recombining sequences are placed in inverse order at widely separated sites in the circular chromosome of Salmonella typhimurium. Recombination can reassort markers between these repeated sequences (double recombination and apparent gene conversion),or can exchange flanking sequences, leading to inversion of the chromosome segment between the recombining sequences. Sincetwo recombinant products remainin the chromosomeof a recombinant with an inversion, one can, in principle, approachthe capability of tetrad analysis. Using this system, the following observations have been made. (a) When long sequences(40 kb) recombine, conversion frequently accompanies exchange of flanking sequences.(b) When short sequences(5 kb) recombine, conversion rarely accompanies exchange of flanks.(c) Both recA and recB mutations eliminate inversion formation. (d) The frequency of exchanges between short repeats is more sensitive to the distance separating the recombiningsequences in thechromosome. The results are presented with the assumptionthat inversions occur by simpleinteraction oftwo sequences in the samecircular chromosome. In an appendix we discuss mechanistically more complex possibilities, some of which 11 could also apply to standard fungalsystems. HE study of homologous recombination in fungi mer following cutting at cos. Thus, in these standard T has benefited from the recovery and character- recombination systems, substrates are provided with ization of both products of a single meiotic recombi- features that are unlikely to be present in “normal” nation event associated as haploid spores in an ascus uninfected (or unmated) cells but are likely to influ- (reviewed by STAHL1979; PETES, MALONEand SYM- ence the recombination process. INGTON 1991). In bacteria, the haploid nature of the Transfer of chromosome fragments (mating, trans- genome makesit difficult to recover both productsof duction, transformation) appears to rare be for enteric an exchange. In most bacterial recombination systems, bacteria in nature (OCHMANand SELANDER, 1984; a single selected recombinant type is recovered; the MILKMAN and BRIDGES,1993). Inview of this,it seems second, unselected, product of the same exchange is likely that therecombination systems of these bacteria lost. Exceptions to this ruleare phage crosses, in which evolved, not for mating, but to perform the internal both recombinationproducts may berepresented sister-strand exchanges needed for repair and dupli- among the progeny of a single burst (SARTHYand cation formation, processes for which the initiating MESELSON1976). However, since multiple rounds of substrates (nicks or breaks) must be generated endog- phage recombination occur,the origin of complemen- enously. This endogenous recombination is like that tary recombinant types remains uncertain. Much in- in eukaryotic cells, which also uses internal substrates formation on bacterial recombination has been gath- and does not normally deal with injected fragments. ered from conjugation and transduction experiments, The sexual recombination systems used to study bac- both of which involve interaction between a linear terial recombination may focus on events that differ donated molecule (with recombinogenicdouble- in important respects from the events for which the stranded ends) and the uninterrupted circular bacte- recombination system of bacteria evolved. rial chromosome. Double-stranded endsare also pres- If the bacterialrecombination system normally ent in the infecting lambda phage genome prior to acts primarily on sequences within a single chromo- circularization and in the replicated lambda concata- some, it has the potential to cause chromosome re- ’ Present address: National Institute of Mental Health, Laboratory of arrangements. Selective forces acting to conserve the Molecular Biology, Building 36, Room 1B-08. Bethesda, Maryland 20892. genetic mapmay have secondarily shaped the bacterial Genetics 136 27-39 (January, 1994) A. M. Segall and J. R. Roth / FIGURE1 .-Recombination events be- Full Half tween inversely oriented homologous se- exchange recombinationDouble Conversion exchange quences in the same circularbacterial chromosome. Thick lines represent ho- mologous sequences. Triangles represent insertions of drug resistance elements within the homologous sequences. Note that inversion requires a full exchange, 1 C defined as an event in which both pairs of flanking sequences are rejoined. y lac'+ \ lac+ + Inv c C Inversion No Inversion No Inversion Lethal recombination mechanisms so as to minimize inver- reflect a paucity of sites for initiating or resolving the sion formation (which destabilizes the map), while required full exchange (SECALL,MAHAN and ROTH permitting the selectively valuable repair and dupli- 1988; L. MIESEL, A.M. SECALLand J. R. ROTH, cation processes (ANDERSONand ROTH 1977; TLSTY, manuscript submitted). ALBERTINIand MILLER 1984; SONTIand ROTH1989). Direct order repeats can recombine to form dupli- We have previously described a recombination de- cations regardless of the chromosomal position of the tection system in which all the recombining sequences recombining sequences (ANDERSONand ROTH 198 1). are located within the circular bacterial chromosome These internal recombinantscan arise by sister strand (SECALL,MAHAN and ROTH 1988).In this system, exchanges in which either one pair of flanking se- initiatingsubstrates for recombination (nicks or quences is rejoined (half exchange) or by exchange of breaks) must begenerated endogenously. Two ho- both flanks (MAHANand ROTH 1988, 1989) (see Fig- mologous sequences, containing distinct genetic mark- ure 4). The unrestricted formation of duplications ers, areplaced in inverse orientation at separatedsites and the fact that they can form by a half exchange in thechromosome (adapted from KONRAD 1969, suggests that they might form by a different mecha- 1977). Recombination betweenthese inverse-order nism from that leading to inversion. sequences can have either of two outcomes (see Figure We have tested this prediction by observing the 1). Some recombinants carry an inversion of the chro- dependence of various internal recombination events mosomal segment between the sites of the recombin- on the RecBCD enzyme, which is required for the ing sequences. These could be caused by a single full major, reciprocal recombination pathway in Esche- crossover event. Other recombinants show reassort- richia coli (CLARK 1973; THALERand STAHL 1988; ment of genetic markers between the repeated se- KINGand RICHARDSON1986; TAKAHASHIet al. 1992). quences but have not acquired an inversion. Some of We show here that mutations in the recB gene elimi- these retain all genetic markers and may have been nate inversion formation, but have little effect on the caused by double crossovers; others have lost one of frequency of duplications. We suggest that this differ- the genetic markers and appearto have resulted from ence reflectsthe fact that inversion formation formally gene conversion events. requires a full exchange (both flanks rejoined), which Only a subset of the many chromosomal intervals can beprovided only by the RecBCD pathway. In tested are permissive for inversion. Inverse repeats contrast, duplications can be generated either by a placed at nonpermissive sites can exchange informa- full exchange (using RecBCD) or by a half-exchange tion(apparent double exchanges and conversions), event (RecBCD-independent), in which only one pair but do not generate a recoverable inversion (KON- of flanking sequences is exchanged and two broken RAD 1969, 1977; ZIEG and KUSHNER1978; REBOLLO, ends are generated. FRANCOISand LOUARN 1988; SEGALL, MAHANand Furthermore, we examine the products of recom- ROTH 1988; SECALLand ROTH 1989). The nonper- bination between inversely oriented sequences located missivity of recombination at some sites is not due to at sites that permit inversion of the intervening chro- lethality of the final inversion product,but could mosomal segment (permissive sites). We have assessed Half-Tetrad Analysis in Bacteria 29 TABLE 1 Strain list TT12262 trp(ED)2490::MudA(lacZ478::Tn5)hisD9953::MudA(lacZ95O::TnlO) srl-201 (inverse order MudA insertions) TT12265 trp(E,D)2484::MudA(lacZ478::Tn5)hisD9953::MudA(lacZ950::TnlO) srl-201 (direct order MudA insertions) TT13420 thr-469::MudA(lacZ478::Tn5)pyrB2691::MudA(lacZ95O::TnlO) srZ-201 (inverse order MudA insertions) TT13422 pyrC2688::MudA(lacZ478::Tn5)hisD9953MudA(lacZ95O::TnlO) sd-201 (inverse order MudA insertions) TT13425 pncB220::MudA(lacZ478::Tn5)hisD9953::MudA(lacZ950::TnlO) srl-201 (inverse order MudA insertions) TT13427 trp(ED)2490::MudA hisD9953::MudJ(lacZ951:TnlO) (inverse order MudA and MudJ insertions) TTl3428 trp(EDj2490::MudA hisD9953MudJ(lacZ950::TnlO)(inverse
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