Transduction of Merodiploidy: Induced Duplication of Recipient Genes

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1 JOURNAL OF BACTERIOLOGY, July 1969, p Vol. 99, No. I Copyright 1969 American Society for Microbiology Printed in U.S.A. Transduction of Merodiploidy: Induced Duplication of Recipient Genes C. W. HILL,' DINAH SCHIFFER, AND PAUL BERG Department of Biochemistry, Stanford University Schtool of Medicine, Palo Alto, California Received for publication 12 May 1969 Escherichia coli PB160, which carries a tandem duplication with the gene order metb+ argh- su,59+ thi+: metb+ argh+ su,59- thi+, was used to study the mechanism of P1 transduction of genes in the duplicated region. Transduction of the su,s9+ allele contained within the duplicated segment yields two kinds of su159+ recombinants: 91 % are haploid SU159+ and 9% are su159+/1us59- merodiploids. The duplication in these merodiploid transductants includes the metb locus; however, both copies of the metb locus usually are derived from the recipient. Thus, the requirements for transduction of the "condition of merodiploidy" appear to be the cotransduction of the repeat point (the region where the duplication begins to repeat itself) and, of course, the selected marker (in this case su,59+). A mechanism whereby two recipient chromosomes interact with the transduced "repeat point" region to regenerate the tandem duplication is implicated. It appears that a duplication much larger than the quantity of genetic material carried by a P1 phage can be produced in a transductant. Recent experiments have revealed that the genetic instability associated with certain missense suppressor strains of Escherichia coli is attributable to a duplication of genetic material (3). One such strain, PB160, was heterozygous for the linked sui59 and argh loci. Experimental findings concerning this strain could best be explained by assuming that it has a tandem duplication of the argh sui59 region. Evidence to be presented below strengthens the previous conclusions that the merodiploid is metb+ argh- SU+159 thi+ : metb+ argh+ SU-159 thi+. Transduction of the su+159 allele from this merodiploid to a su-159 recipient yields two kinds of su+ recombinants: 91 % are haploid su+159 and 9% are su+159 / su-59 merodiploids (3). The merodiploidy of these SU+159 / SU-159 transductants includes the metb locus. In this communication we show that transduction of this condition of merodiploidy to a metbt recipient causes a duplication of the resident metth allele, rather than the addition of one of the donor metb+ alleles to the recipient genome. MATERIALS AND METHODS Bacteria. The derivation of the E. coli K-12 strains used has been described previously (3), except that PB174 (trpa36 metbt argh+ su-,59) was prepared by using a phage P1 lysate of PB153 (mettb argh+) to I Present address: Department of Biological Chemistry, Pennsylvania State University, Milton S. Hershey Medical Center, Hershey, Pa transduce PB154 (metb+ argh") to Arg+ and screening for a Met- cotransductant. To simplify the designations, only pertinent genetic markers will be listed with the strain numbers in the text. Based in part on evidence described previously (3) and in part on evidence presented here, the gene order in the duplicated region of PB160 is metb+ arghf su+1,9 thi+ : metb+ argh+ SU-159 tht+. Bacteriophage. The phage T4 missense mutant, T4s3, grows only on E. coli strains which carry the specific suppressor of trpa36 (4). Genetic symbols and abbreviations. Genetic symbols refer to loci concerned with the biosynthesis of tryptophan (trp), methionine (met), arginine (arg), and thiamine (thi), and specific allele designations are as used by Taylor and Trotter (5); su+159 refers to a trpa36 specific suppressor, and su-,59 refers to the wild-type allele of this suppressor gene. Trp+, Arg+, Met+, and Thi+ refer to tryptophan-, arginine-, methionine-, and thiamine-independent phenotypes, respectively, whereas Trp-, Arg-, Met-, and Thirefer to the respective auxotrophic phenotypes. The locus for su,,s maps between argh and thi (3); other gene orders are as given by Taylor and Trotter (5). The symbol ":" (as argh- su+,,,: argh+ SU-169) is used to designate that the duplicated linkage groups are tandemly inserted in the bacterial chromosome with the gene order designated, whereas the symbol "/" (as argh- su+159 / argh+ su-15,) indicates the presence of a duplication without specifying the relative positions of the linkage groups. Media and procedures. Media and procedures for transduction and for the testing of clones for growth requirements were as described previously (3). 274

2 VOL. 99, 1969 INDUCED DUPLICATION OF RECIPIENT GENES RESULTS Strain PB160 carries a genetic duplication which includes the metb as well as argh and su,59 loci. Previous studies of the unstable suppressor strain, PB160, indicated that it was a merodiploid with the genotype argh- su+159 : argh+ su-159, and that the duplication was integrated in tandem within the bacterial chromosome (3). Evidence for these conclusions included the following: the recessive argh- allele could be recovered either by segregation or by cotransduction with the linked su+159 allele, demonstrating the involvement of a genetic duplication; duplicated loci are cotransferred in interrupted-mating experiments, showing that both copies are integrated within the chromosome; the segregation pattern of duplicated markers is that predicted by a mechanism of segregation whereby the copies of a tandem duplication pair, and recombine, causing the elimination of one set of the duplicated genes (see also Table 2 below); and segregation of markers is blocked by the presence of the recombinationdefective rec-56 allele. Although segregants of PB160 generally become haploid, occasional segregants, such as PB161, are Arg-, but remain heterozygous for the su159 locus (3). PB161 is concluded to have become homozygous at the argh locus (arghsu+,59 : argh- su-159), since transduction of this strain to Arg+ restores the heterozygosity of the argh- locus: the recessive argh- allele can again be recovered by segregation (see Table 2) and by cotransduction (unpublished data). If the metb locus is also included in the duplication, so that these strains are metb+ argh su+159 : metb+ argh- su-159, two predictions can be made. (i) When a metth argh+ donor is used to transduce PB161 (argh- / argh-) to Arg+, the cotransduction of the Met- phenotype should be sharply reduced from the normal 24%, since, even if metb is cotransduced with argh+, the transductant will be phenotypically Met+ (metb argh+ / metb+ argh-). (ii) Approximately 24% of these Arg+ transductants should carry a recessive metb allele which can be recovered by segregation or by cotransduction with a linked dominant marker. To verify these predictions, a phage P1 lysate of PB153 (metb argh+) was used to infect PB161 (argh" su+159 : arghi su-159) and Arg+, Trp+ transductants were selected (Table 1, experiment b). In agreement with the first prediction, none of 34 transductants became Met-, whereas the cotransduction frequency of argh+ and metb between haploid strains was found to be 24% (Table 1, experiment a). Phage P1 lysates of 26 of these Argt, Met+ transductants from Table 1, experiment b, were used to transduce a metb+ argh- recipient to Arg+, and these transductants were scored for their methionine requirement. In keeping with the second prediction, the metbc allele could be recovered in 5 (19%) of the 26 cases, indicating that these 5 had been metbc argh+ / metb+ argh- merodiploids. For two of these five (PB175 is an example), the transduction experiments showed the presence of a metbr argh+ SU+159 linkage group, suggesting a gene order of metb7 argh+ SU+159: metb+ arghsu-159. The other three (PB176 is an example) exhibited no linkage of argh+ and su+159, indicating the other possible gene order: metb+ arghsu+159 : mettb argh+ SU-159. Segregation patterns for these metb+ / metbmerodiploids are consistent with the gene orders suggested by the transduction experiments. As discussed previously (3), one of the consequences of the tandem duplication structure is a high frequency of segregation of markers within the duplication. This presumably results from pairing between homologous regions within the duplication, followed by a recombinational event and elimination of one set of the duplicated genes (1, 3; Fig. 1). Trp-segregants (actuallysu-159) were collectedfromtheputativemerodiploidtransductants, PB175 (metb argh+ SU+159: metb+ argh- su-159) and PB176 (metb+ argh- su+159 : metb argh+ SU-1159) from above, and scored for their Met, Arg phenotypes (Table 2). Each of the four possible Met, Arg phenotypes, together with the position of the crossover event needed to produce that type of segregant, is indicated in Table 2: of the four possible phenotypes, the ones requiring single crossovers should occur more frequently than the one needing triple crossovers. The results are clearly consistent with the assigned gene orders for PB175 and PB176 indicated above. From these experiments we concluded that the duplication present in PB161, and therefore in its pro- \.N_ metb argh su.-~ \ // I a V b c I'.I/\ I, metb argh su FIG. 1. Possible scheme for segregation from a tandem duplication. The segregation may involve a single crossover between the copies of the duplication within a single chromosome, resulting in the elimination ofone complete copy as a small circle; an alternative scheme, in which a single crossover occurs between the left hand copy of one chromosome and the right hand copy of another, predicts the same distribution of segregant phenotypes.

3 276 HILL, SCHIFFER, AND BERG J. BAcEmuoL. TABLE 1. Phenotype frequencies of haploid and merodiploid transductants- Expt Donor Recipient Selected markers No. of Occurrence trans- noccurreneyof ductant unselected phenotypes a PB153 (metb argh+) PB154 (metb; arghl) argh (24%) Metb PB153 (metbi argh+ PB161 (metb+ arghi argh+, sul5g (0%) Met- SU159-) SUiss+: metba arghi SU1i97) c PB160 (metb+ arghi PB174 (metbt argh+ (su559+ / suls59) 27 2 (7%) Met+ SU16s+: metb+ argh+ SUIss9) 25 (93%) Met7 SU15i ) d PB162 (argh+ su159+ PB155 (arghi SUisi- SU15g (45%) Thi+ thi+) thi-) 44 (51%) Arg+ e PB160 (argh" Sui5g PB155 (arghi sui597 (SU16s+ / su16s7) (100%7o) Thi+ thia : argh+ SUi597 th ) 0 (0%0) Arg+ thi+) Selection was done on minimal glucose plates supplemented with (a) methionine and tryptophan; (b) methionine; (c) 0.1% Casamino Acids and (100pg/ml, each) adenosine, guanosine, cytidine, and uridine; (d) 0.1% Casamino Acids and thiamine; (e) same as c, but with thiamine. Tryptophan auxotrophs will not grow with Casamino Acids unless tryptophan is also supplied. The presence of the SUi59+ mutation in a trpa36 strain produces a tryptophan-independent phenotype. Haploid su159+ mutants are severely restricted in the presence of 0.1% Casamino Acids and the four common ribonucleosides, whereas (su159+ / SU1697) partial heterozygotes are not (3). Transductant colonies were scored for unselected markers without purification. Phenotype of suj5&9 segregants TABLE 2. Segregation of markers in merodiploids" Region of required recombination if gene order is: Distribution of observed phenotypes meit argh+ suu16+: metb+ metb+ argh- suu,+ : metb PB175 PB176 argh" su,,i- (PB175) argh+ su,,- (PB176) Met+, Arg- a c Met, Arg- b a, b, and c 22 1 Met-, Arg+ c a Met+, Arg+ a, b, and c b 2 28 a After at least three successive single-colony isolations, cultures of PB175 and PB176 were diluted to 104 cells/ml in minimal-glucose medium supplemented with 0.1% Casamino Acids and grown to about 10' cells/ml (log phase), at which time they were spread on agar plates of the same medium containing 7.5 pug of tryptophan per plate. On these limiting tryptophan plates, Trp- (su1597) segregants produce very small, flat colonies which are easily distinguishable from the large colonies produced by the nonsegregants. All of the Trp- segregants detected were tested for their Met and Arg phenotypes. For each strain, the data from several clonally independent experiments are pooled. Fifty-one of 1,468 cells from PB175 and 71 of2,346 cells from PB176 were Trp-. The regions of recombination are specified in Fig. 1. genitor PB160, includes the metb locus, and that the gene order of PB160 is metb+ argh SU+159 metb+ argh+ su-1g9. Transduction of merodiploidy: induced duplication of the recipient metb allele. The PB160 tandem duplication can be transduced by phage P1. Transduction of the SU+159 allele from this SU+1s5 / SU-6g merodiploid results in about 91% su+159haploid transductants and 9% su+s59 / su- 59 merodiploid transductants (3). This frequent occurrence of merodiploid transductants is dependent on the merodiploid state of the donor strain, since only 0.1% of the total transductants are su+l59 / SU-15 merodiploids if a haploid su+5(s strain is used as donor (3). One possible mechanism for the transduction and integration of a tandem duplication is one in which the transduced material pairs and recombines with a single chromosome of the recipient. This model is shown formally in Fig. 2A. Pairing occurs between the recipient chromosome and the extreme ends of the duplication on the transduced piece; an unpaired loop is formed which contains exactly one copy of each duplicated gene and the point at which the repeat begins. A recombinational event on each side of the loop

4 VOL. 99, 1969 INDUCED DUPLICATION OF RECIPIENT GENES 277 d^o A. c( ) m obs Mcd n B. M A C. STEP M A C STEP 11and BC DN B )MAB8 c d ~-0 a bc n N ) M ABC dp0oa CDN d CIB ) MA_ CtoaaCaN FIG. 2. Models for the transduction of a tandem duplication. Capital letters represent recipient genes; lower case letters represent donor genes. The circle diagrammatically represents the point at which the repeat begins. Model A requires that the transductant receive at least one copy ofeach duplicated gene from the donor. Models B and C make no such requirement; in the examples illustrated, both copies of genes B and C are derivedfrom the recipient. In the particular case of strain PBI60, genes b, c, and d correspond to metb, argh, and sui59, respectively; gene a would represent some undetermined gene very close to the left hand end of the duplication. produces a transductant which has a duplication of the same extent as the donor. Such a model requires that the transductant receive at least one copy of each duplicated marker from the donor. The following experiments show that this requirement is not met when the merodiploid character of PB160 is transduced to a haploid recipient. In these experiments, advantage was taken of the observation that su+159 / su-159 merodiploids grow well on minimal glucose-agar plates supplemented with 0.1% Casamino Acids and 100,ug (per ml) of each of the four common ribonucleosides, adenosine, guanosine, cytidine, and uridine, whereas haploid su+159 strains are severely restricted on this medium (3). A phage Pl lysate of PB160 (metb+ argh- su+159 : metb+ argh+ su-159) was used to transduce the suppressor to a mete- argh+ su-159 strain; tryptophan-independent transductants were selected on the above agar plates to ensure isolation of the su+159 / su-159 merodiploids (Table 1, experiment c). Twentyseven colonies appeared on the transduction plates, all of which were permissive for the phage T4 missense mutant, T4s3 (4), indicating they had all received su+159. Two of these 27 were Met+ cotransductants, and these are either metb+ su+,59 / metb- su-,5g or metb- su+159 / metfb su-,59 merodiploids since they simultaneously lose the metb+ and su+,59 markers by segregation. The remaining 25 su+l59 / su-159 transductants were Met-; of 6 that were examined further, all were shown to be metth su+159 / metb- su-159 merodiploids as follows. Phage P1 grown on a metb+ su-159 strain transduced each of these six merodiploids to Met+. Even after three successive single-colony isolations, the transductants still gave rise to su-159 (Trp-) segregants, many of which were also Met-. This shows that the Met+ transductants retained the recessive mettb allele. Thus, it appears that, when a lysate of the metby su+59 : metb+ su-159 merodiploid, PB160, is used to transduce the suppressor heterozygosity to a mettb su-5g recipient, the merodiploid transductants very frequently have the genotype metbc su+5sg / metb- su-159; that is, the resident metb allele has been duplicated during the transduction. DISCUSSION The fact that the large majority of the merodiploid transductantsdescribed abovederived both copies of the metb gene from the recipient cannot be accounted for by the mechanism for duplication transduction illustrated in Fig. 2A. The frequent appearance of these transductants can be explained by a mechanism of the type proposed by Campbell (2) and illustrated in Fig. 2B or 2C. In Fig. 2B the donor deoxyribonucleic acid (DNA) recombines on each side of the repeat point with each of two different recipient chromosomes (from separate nuclei or arms of the replicating chromosomes). If a third recombinational event occurs between the recipient chromosomes, the production of a duplication in the recipient, identical in extent to that of the donor, will occur. The model diagrammed in Fig. 2C is similar except that the donor DNA containing the repeat point recombines initially with a single chromosome to generate a small circle (Fig. 2C, step I) which in turn can integrate into a second chromosome to generate the tandem duplication (Fig. 2C, step II). An alternative explanation, that the merodiploid transductants initially were metb+ / metb-, but became mete- / metb- homozygotes by a subsequent recombinational event, is thought highly unlikely for two reasons. First, the original su+169 / su-m9 merodiploid transductant colonies were scored directly for their methionine requirement, and the presence of even a few metb+ / metbc cells in a largely mete- / metb- colony would have been detected. Second, several established metb+ / metb- merodiploids (such as

5 278 HILL, SCHIFFER, AND BERG J. BACrERioL. PB175 and PB176) have been handled extensively and conversion to metb / metb has been found to be a rare event. All three models in Fig. 2 require that the repeat point and the selected marker be cotransduced when a merodiploid transductant is produced. It would be expected that any markers between the repeat point and the selected marker would be cotransduced with a very high frequency. This was found to be the situation with the thi+ marker in PB160. When a lysate of PB160 was used to transduce SU+l59 to a SU-1 g thirecipient, all of 42 su+159 / su-159 transductants received the thi+ allele from the donor (Table 1, experiment e). This 100% cotransduction of su+159 and thi+ is markedly different from the 45% normally observed with haploid donors (Table 1, experiment d). Moreover, all (25 of 25) of the su-59 segregants from four of these SU+159 / su-159 transductants were Thi-. Transduction of the tandemly duplicated character by phage P1 requires only that the selected marker and the repeat point be cotransduced; if neither copy of a duplicated gene lies between the selected marker and the repeat point, neither need be cotransduced. Thus, as illustrated in Fig. 2B and 2C, a duplication much larger than the quantity of genetic material carried by the P1 phage can be produced in a transductant, the only restriction being that the selected marker and the repeat point be sufficiently closely linked to be accommodated by the P1 phage. In fact, two lines of evidence indicate that even the length of DNA in PB160 which includes su+159, the repeat point, and argh+ is too large to be carried by a P1 phage. First, the su+l59 and argh+ markers of PB160 (argh- SU+159: argh+ su-159) are rarely (0.3%) cotransduced (3). That this frequency is not zero could be explained by the presence in the culture of occasional cells which have undergone some sort of exchange to put argh+ and su+1s9 on the same copy of the duplication. Second, if the frequencies of the various types of segregants in the experiments described in Table 2 reflect the physical distances between markers, region a is larger than region b and c combined. The map of Taylor and Trotter (5) gives the distance between metb and argh (region b) as 0.9 min. The total length of a single copy of the duplication must then be greater than 3 min, a distance too long to be accommodated by a phage P1 particle (5). In summary, we have shown that the "condition of merodiploidy" may be transduced by phage P1 from one strain of E. coli to another, even when the amount of genetic material duplicated is large relative to that normally carried by a P1 transducing particle. This phenomenon is most readily explained by mechanisms such as those illustrated in Fig. 2B or 2C, whereby two recipient chromosomes interact with the transduced repeat point region to regenerate the tandem duplication. ACKNOWLEDGMENTS We thank Charles Yanofsky, Alan Campbell, and John Clark for helpful criticisms of this work. This investigation was supported by Public Health Service research grant GM from the National Institute of General Medical Sciences. C.W.H. gratefully acknowledges support by a postdoctoral fellowship from the American Cancer Society. LITERATURE CITED 1. Campbell, A Segregants from lysogenic heterogenotes carrying recombinant lambda prophages. Virology 20: Campbell, A The steric effect in lysogenization by bacteriophage lambda. I. Lysogenization of a partially diploid strain of Escherichia coil K12. Virology 27: Hill, C. W., J. Foulds, L. Soll, and P. Berg Instability of a missense suppressor resulting from a duplication of genetic material. J. Mol. Biol. 39: Reid, P., and P. Berg T4 bacteriophage mutants suppressible by a missense suppressor which inserts glycine in place of arginine for the codon AGA. J. Virol. 2: Taylor, A. L., and C. D. Trotter Revised linkage map of Escherichia col. Bacteriol Rev. 31: