recombination in the vicinity of insertions of …dk3225 r2224(tn5-wt)ql900(tn5-132) mx4(dk2777) x...

11
Copyright 0 1983 by the Genetics Society of America RECOMBINATION IN THE VICINITY OF INSERTIONS OF TRANSPOSON Tn5 IN MYXOCOCCUS XANTHUS ERICA SODERGREN,' YVONNE CHENG, LEON AVERY AND DALE KAISER Department .f Biochemistry, Stanford University, Stanford, Calijiornia 94305 Manuscript received March 25, 1983 Revised copy accepted June 7, 1983 ABSTRACT To test genetic recombination in the vicinity of insertions of the transposon Tn5, crosses were performed by transduction between M. xunthw strains car- rying different insertions of Tn5. One member of each pair carried resistance to kanamycin (Tn5-Km); the other carried resistance to tetracycline (Tn5-Tc). The distance between each pair of Tn5 insertions was also measured by re- striction mapping. The physical distance corresponding to each recombination frequency was calculated from the transductional linkage and compared with distance on the restriction map. A good correspondence between the two meas- ures of distance was obtained for a pair of Tn5 insertions near the cglB locus and for another pair near the mgl locus. Correspondence between the two measurements of distance, the observed allelic behavior of Tn5-Km and Tn5- Tc at the same locus and the finding of the same frequencies of recombinants in reciprocal crosses implied that recombination in the vicinity of T n 5 was normal. ESPITE their ability to move to new chromosomal sites, bacterial transpo- D sons are generally assumed to be well-behaved genetic markers in crosses (KLECKNER, ROTH and B~TSTEIN 1977). The evidence that transposon inser- tions behave in crosses as point mutations comes from three types of experi- ments. (1) When the insertion of a drug-resistant transposon destroys the func- tion of a gene, the deficiency genotype and the drug resistance are completely linked (KLECKNER et al. 1975; BERG 1977). (2) Three point tests with a transposon at one point give the expected order (KLECKNER, ROTH and BOT- STEIN 1977). (3) In crosses between members of a set of overlapping deletions and a transposon insertion, the transposon maps to a single, deletion-defined interval (HOPPE et al., 1979). The behavior of transposon insertions as point mutations can also be tested using two versions of Tn5, the wild type that carries kanamycin resistance (Km) and a new version that carries tetracycline resistance (Tc) instead (ROTH- STEIN et al. 1981; BERG et al. 1981). A pair of different Tn5 insertions can be marked with Km and Tc and a cross between them performed to obtain the recombination distance between the two sites of transposon insertion. The physical distance between the same two insertions can be measured by digestion ' Present address: National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21701. Genetics 105: 281-291 October, 1983.

Upload: others

Post on 14-Mar-2021

5 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: RECOMBINATION IN THE VICINITY OF INSERTIONS OF …DK3225 R2224(Tn5-wt)Ql900(Tn5-132) Mx4(DK2777) X DK1850 Each independent insertion of the transposon Tn5 is identified by an R number

Copyright 0 1983 by the Genetics Society of America

RECOMBINATION IN THE VICINITY OF INSERTIONS OF TRANSPOSON T n 5 IN MYXOCOCCUS XANTHUS

ERICA SODERGREN,' YVONNE CHENG, LEON AVERY A N D DALE KAISER

Department .f Biochemistry, Stanford University, Stanford, Calijiornia 94305

Manuscript received March 25, 1983 Revised copy accepted June 7, 1983

ABSTRACT

To test genetic recombination in the vicinity of insertions of the transposon Tn5, crosses were performed by transduction between M. xunthw strains car- rying different insertions of Tn5. One member of each pair carried resistance to kanamycin (Tn5-Km); the other carried resistance to tetracycline (Tn5-Tc). The distance between each pair of Tn5 insertions was also measured by re- striction mapping. The physical distance corresponding to each recombination frequency was calculated from the transductional linkage and compared with distance on the restriction map. A good correspondence between the two meas- ures of distance was obtained for a pair of Tn5 insertions near the cglB locus and for another pair near the mgl locus. Correspondence between the two measurements of distance, the observed allelic behavior of Tn5-Km and Tn5- Tc at the same locus and the finding of the same frequencies of recombinants in reciprocal crosses implied that recombination in the vicinity of Tn5 was normal.

ESPITE their ability to move to new chromosomal sites, bacterial transpo- D sons are generally assumed to be well-behaved genetic markers in crosses (KLECKNER, ROTH and B~TSTEIN 1977). The evidence that transposon inser- tions behave in crosses as point mutations comes from three types of experi- ments. (1) When the insertion of a drug-resistant transposon destroys the func- tion of a gene, the deficiency genotype and the drug resistance are completely linked (KLECKNER et al. 1975; BERG 1977). (2) Three point tests with a transposon at one point give the expected order (KLECKNER, ROTH and BOT- STEIN 1977). (3) In crosses between members of a set of overlapping deletions and a transposon insertion, the transposon maps to a single, deletion-defined interval (HOPPE et al., 1979).

The behavior of transposon insertions as point mutations can also be tested using two versions of Tn5, the wild type that carries kanamycin resistance (Km) and a new version that carries tetracycline resistance (Tc) instead (ROTH- STEIN et al. 1981; BERG et al. 1981). A pair of different Tn5 insertions can be marked with Km and Tc and a cross between them performed to obtain the recombination distance between the two sites of transposon insertion. The physical distance between the same two insertions can be measured by digestion

' Present address: National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21701. Genetics 105: 281-291 October, 1983.

Page 2: RECOMBINATION IN THE VICINITY OF INSERTIONS OF …DK3225 R2224(Tn5-wt)Ql900(Tn5-132) Mx4(DK2777) X DK1850 Each independent insertion of the transposon Tn5 is identified by an R number

282 E. SODERGREN ET AL.

with restriction endonucleases and compared with the recombination distance to test the efficiency of recombination in the interval between the two trans- posons. The same set of experiments also tests the allelism of two transposons at the same site as well as the equivalence of reciprocal cotransduction of two transposons at different sites. This paper reports such tests with Tn5 pairs in two different regions of the chromosome of Myxococcus xanthus.

MATERIALS AND METHODS

Strains, inedia and transdurtion: Strains are listed in Table 1. Media and the protocol for trans- duction are described in SODERGREN and KAISER (1 983).

Tn5-I32 and replacement of Tn5 by Tn5-132: Tn5-132 has been constructed by replacing the aminoglycoside phosphotransferase structural gene, which resides on a central BglII fragment of Tn5, with a BglII fragment from T n l 0 that includes the entire tetracycline-resistant gene (ROTH- STEIN et al. 1981; BERG et al. 1981). Tn5-132 has 1.504 of the 1.534 kb of both IS50 units of Tn5 and, thus, retains transposability, which is associated with the IS50 units (AUERSWALD, LUDWIG and SCHALLER 1981; BERG et al . 1981). Tn5-132 (Tc) can replace Tn5-wt (Km) through homol- ogous recombination in both IS50 units, thereby retaining the original chromosomal site of Tn5 insertion (BERG, WEISS and CROSSLAND 1980; AVERY and KAISER 1983). Replacements were car- ried out as described by AVERY and KAISER (1983). After single colony isolation of a Tc’Km” strain, the structure of its DNA in the vicinity of its Tn5 insertion was verified by restriction endonuclease analysis (AVERY and KAISER 1983).

Restrictroil endonuclease aiirilysis of Myxococcus DNA: DNA was obtained as described by KUNER and KAISER (1981). DNA samples were cleaved with restriction enzymes as follows: EcoRI in 50 n l M Tris.HCI (pH 7.4), 100 mM NaCI, 10 mM MgC12, as described by WAHL, PADGETT and STARK (1979); BninHI in 6 mM Tris.HCI (pH 7.6), 50 mM NaCI, 6 mM MgC12, 100 pg/ml of bovine serum albumin (Bethesda Research Laboratories), 7 mM mercaptoethanol; and Hind111 (New Eng- land Biolabs) in 20 mM Tris.HCI (pH 7.4), 60 mM NaCI, 7 mM MgC12, 100 pg/ml of bovine serum albumin, 2 mM dithiothreitol, as described by WAHL, STERN and STARK (1979). The re- sulting DNA fragments were separated according to size by electrophoresis through a 0.4% agarose gel (MCDONELL, SIMON and STUDIER 1977) and transferred to nitrocellulose paper according to the procedure of SOUTHERN (1975) as modified and described by WAHL, STERN and STARK (1979).

To test for Tn5 sequences, plasmid ColEl::Tn5 DNA (KUNER and KAISER 1981) was radioac- tively labeled with [a-’*P]dCTP (Amersham Corporation) by nick translation using DNA polym- erase I (RIBGY et al . 1977). This radioactively labeled DNA was hybridized to nitrocellulose-bound DNA fragments according to the procedure of DAVIS, BOTSTEIN and ROTH (1980). Hybridizations were carried out in 0.02% (w/v) bovine serum albumin, Ficoll M , 400,000 and polyvinyl pyrroli- done, 0.9 M NaCI, 50 mM NaHZP04, 5 mM EDTA, pH 7, 50% formamide, 0.1% sodium dodecyl sulfate, and 500 pg/ml of sonicated, boiled calf thymus DNA. Following hybridization the nitro- cellulose filters were washed in 50% formamide, 0.9 M NaCI, 50 mM NaHZP04, 5 mM EDTA, 0.3% SDS at 37”.

Size standards for DNA were obtained by digesting phage XDNA with HindIII endonuclease and separately with XhoI endonuclease. HindIII cleavage generates DNA fragments of 23.7, 9.5, 6.7, 4.3, 2.3 and 2.0 kb from XDNA (DAVIS, BOTSTEIN and ROTH 1980). XhoI generates 34- and 15-kb fragments. A standard of 28 kb was generated by the annealing of the two Hind111 end fragments (23.7 + 4.3) through the X cohesive ends; whole XDNA is the standard for 49 kb. A small amount of XDNA was also added to the ColEI::Tn5 DNA before nick translation (previous paragraph) to allow radioautographic visualization of the XDNA size standards. M. xanthus contains no sequence that hybridizes with X or ColEl.

RESULTS

Physical mapping of pairs of Tn5 insertions: Tn5 insertions ill902 and Q1933 are linked to the mgl locus by cotransduction (SODERGREN and KAISER 1983).

Page 3: RECOMBINATION IN THE VICINITY OF INSERTIONS OF …DK3225 R2224(Tn5-wt)Ql900(Tn5-132) Mx4(DK2777) X DK1850 Each independent insertion of the transposon Tn5 is identified by an R number

RECOMBINATION NEAR T N 5 283

TABLE 1

Bucterial strains

Strain Tn5 elements" Reference or source'

DK 1850 Q1900(Tn5-132) Replacement of Tn5-wt by Tn5-132 in DK1900

DKl85 1 Q1902(Tn5-132) Replacement of Tn5-wt by Tn5-132 in DK1902

DKl853 Q1933(Tn5-132) Replacement of Tn5-wt by Tn5-132 in DK1933

DK1870 R2224(Tn5-132) Replacement of Tn5-wt by Tn5-132 in DK2777

DK1871 Q1900(Tn5-wt)Q2224(Tn5-132) Mx4(DK1900) X DK1870 DK1880 Q 1933(Tn5-wt)Q 1902(Tn5-132) Mx8(DK1933) X DK1851 DK1883 Q 1902(Tn5-wt)R 1933(Tn5-I 32) Mx8(DK1902) X DK1853 DK1900 R1 gOO(Tn5-wt) Sodergren and Kaiser (1 983) DK1902 R1902(Tn5-wt) Sodergren and Kaiser (1983) DK1933 Ql933(Tn5-wt) Sodergren and Kaiser (1 983) DK2777 Q2224(Tn5-wt) Sodergren and Kaiser (1983) DK3225 R2224(Tn5-wt)Ql900(Tn5-132) Mx4(DK2777) X DK1850

Each independent insertion of the transposon Tn5 is identified by an R number as recom- mended by CAMPBELL et al. (1979)-Q1900, for example, which designates the site of insertion. We have adopted the convention of assigning to the insertion the number of the strain in which the particular insertion was first recognized. In the case of Q1900, strain DK1900 serves as a standard source of the insertion, and the convention facilitates tracing genetic histories. When a particular Tn5 insertion is transferred to another strain through homologous recombination, the insertion site is the same and so Q1900 is still used, although the strain number changes. Thus, Table I has DK3225 which carries R1900.

b T h e description Mx8(DKl933) X DK1851 indicates that a stock of the transducing phage Mx8 grown on DK1933 was used to transduce DK1851.

Both 521902 and 521933 lie in EcoRI restriction fragments of about 33 kb in size (Figure 1, compare lanes 1 and 2). There being no EcoRI site in Tn5 itself (BERG et al. 1975; AUERSWALD, LUDWIG and SCHALLER 1981), the similar electrophoretic mobilities of the two Tn5-containing fragments suggests that 521902 and 521933 reside between the same pair of adjacent EcoRI sites in the Myxococcus chromosome; but this point cannot be established by that similarity because size resolution in the high molecular weight range of the gel is limited. If 521902 and Q1933 are different T n 5 insertions between the same pair of EcoRI sites in the Myxococcus chromosome, then a strain that carries an in- sertion of Tn5-wt at one site and Tn5-132 at the other should yield EcoRI fragments that differ in a predictable way from those of either parent. In contrast to Tn5-wt which has no EcoRI site, the tet segment of Tn5-132 has an EcoRI site near its midpoint (JORGENSEN et al. 1979). Consequently a strain that carried Tn5-132 will yield two T n 5 homologous fragments (4 Figure 1, lanes 3 and 4). The addition of Tn5-wt should increase the size of one of these two by the length of Tn5, provided that both insertions are in the same EcoRI segment of the Myxococcus chromosome. If, on the other hand, 521902 and 521933 are not in the same EcoRI segment, then digestion of DNA from the strain carrying both Tn5 and Tn5-132 with EcoRI should yield three fragments containing T n 5 sequences.

Page 4: RECOMBINATION IN THE VICINITY OF INSERTIONS OF …DK3225 R2224(Tn5-wt)Ql900(Tn5-132) Mx4(DK2777) X DK1850 Each independent insertion of the transposon Tn5 is identified by an R number

284 E. SODERGREN E r AL.

- - - - - .

FIGURE I.-Change in restriction tragment size in the vicinity ot mgf with insertion ot a n additional T n 5 . DNA samples were cleaved with EcoRI restriction endonuclease and electropho- resed in 0.4% agarose, and DNA from t h e gel was hybridized with '*P-labeled ColEl::Tn5 DNA. Lane A: Molecular weight standards. A mixture o f Hind111 and Xhol digested phage X DNA. 1: DK1902, R1902 (Tn5-wt). 2: DK1933, 0 1 9 3 3 (Tn5-wt). 3: DK1853, Q1933 (Tn5-132). 4: DK1851, RI902 (Tn5-132). 5: DK1880, R1902 (Tn5-132) Q1933 (Tn5-wt). 6: DK1883, RI933 (Tn5-132) R1902 (Tn5-wt). 7: DK1853, R1933 (Tn5-132).

To perform the test, two complementary double insertion strains were con- structed: RI902 (Tn5-wt) RI933 (Tn5-132) and RI902 (Tn5-132) RI933 (Tn5- wt). The EcoRI restriction patterns of these two strains and their controls are presented in Figure 1. Both double insertion strains (lanes 5 and 6) yield only

Page 5: RECOMBINATION IN THE VICINITY OF INSERTIONS OF …DK3225 R2224(Tn5-wt)Ql900(Tn5-132) Mx4(DK2777) X DK1850 Each independent insertion of the transposon Tn5 is identified by an R number

RECOMBINATION NEAR T N 5 285

A 1 2 3 4 5 6 X A

-49 -34 -28 -23.7

-1 5

-9.5

- 6.7

- 4.3

FIGURE 2.-Change in restriction fragment size in the vicinity of cglB with insertion of an additional Tn5. DNA samples were cleaved with EroRI restriction endonuclease and electropho- resed in 0.4% agarose, and DNA from the gel was hybridized with '*P-labeled ColEl::Tn5 DNA by the method of SOUTHERN (1975). Lane X: Molecular weight standards. A mixture of Hind111 and Xhol digested phage X DNA. 1: DK1900, R1900 (Tn5-wt). 2: DK2777, R2224 (Tn5-wt). 3: DK1870, R2224 (Tn5-132). 4: DK1871, R2224 (Tn5-132) R1900 (Tn5-wt). 5: DK3225, R1900 (Tn5-132) R2224 (Tn5-wt). 6: DK1850. R1900 (Tn5-132). X: Molecular weight standards. Xhol digested phage X DNA.

two fragments that contain T n 5 sequences. In both cases one is the same size as the smaller fragment of the single insertion; the other is approximately one TnZ length longer than the other fragment of the single insertion. For ex- ample, the longer fragment in Q1902(Tn5-132), shown in lane 4, is 18.5 kb and becomes 24.5 kb in the corresponding double insertion strain Q1902(TnZ- 132)Q1933(Tn5-wt), shown in lane 5. Similarly, the longer fragment in Q1933(Tn5-132), shown in lane 3, is 25 kb and becomes 31 kb in Q1933(Tn5-

Page 6: RECOMBINATION IN THE VICINITY OF INSERTIONS OF …DK3225 R2224(Tn5-wt)Ql900(Tn5-132) Mx4(DK2777) X DK1850 Each independent insertion of the transposon Tn5 is identified by an R number

286 E. SODERGREN ET AL.

132)Q1902(Tn5-wt), shown in lane 6. Thus, Q1902 and Q1933 do indeed lie between the same pair of adjacent EcoRI sites.

These data yield a unique restriction map shown in Figure 3A. Results of a

RR 1933 1902 B

BL RL v/ R I I 1

- 5.3- 0.05 - 22

11 - - -11 -

11.5- 16

22

1900 VNBR R R

I I I

0,l -3- 12-

17 - 19

12- 29

FIGURE 3.-Restriction maps of (A): the Q1933-Ql902 region and (B): the 611900-Q2224 re- gion. Distances in kilobase pairs (kb). BL and RL represent the first BamHI and first EcoRI sites in Myxococcus chromosomal DNA on the left and adjacent to the sites of insertion of Q2224 and Q1900. Similarly, BR and RR are the first sites to the right. For the insertions of Tn5, distances are calculated to the point of insertion, taking the length of whole Tn5 as 5.7 kb (AUERSWALD, LUDWIG and SCHALLER 1981), the distance between the central BamHI site and the end of Tn5 as 2.85 kb UORCENSEN, ROTHSTEIN and REZNIKOFF 1979) and the distance between the central EcoRI site of Tn5 tet and the end as 2.8 * 0.2 kb. The error limits in the last case reflect the fact that the EcoRI site is 3 kb from one end of Tn5 and 2.7 kb from the other, and the orientation is unknown UORGENSEN et a l . 1979). From panel (A) there are four independent estimates of the 01933 to Q1902 distance. Measured from BL, the distance is 22 - 11 = 11 kb; from RL 16 - 5.3 = 10.7 kb; from BR 11 - 0 = 11 kb; from RR 22 - 11.5 = 10.5 kb. From panel (B) there are four independent estimates of the Q2224 to 611 900 distance. From restriction site BL the distance is 29 - 12 = 17 kb; relative to BR it is 17 - 0.1 = 17 kb; relative to RL it is 19 - 3 = 16 kb; relative to R R it is 28 - 12 = 16 kb.

Page 7: RECOMBINATION IN THE VICINITY OF INSERTIONS OF …DK3225 R2224(Tn5-wt)Ql900(Tn5-132) Mx4(DK2777) X DK1850 Each independent insertion of the transposon Tn5 is identified by an R number

RECOMBINATION NEAR T N 5 287

restriction analysis with BainHI restriction endonuclease, which cuts once near the center of Tn5-wt but not at all in Tn5-132, are also shown in the figure. Four independent estimates of the distance between the insertion sites of 01933 and 01902 can be computed from this map (Figure 3, legend); their average is 10.8 kb. The range of the four estimates is +1 kb which includes an uncertainty in fragment length due to the finite width of the gel bands,

A parallel analysis was carried out with a pair of T n 5 insertions at 01900 and 02224, which both cotransduce with cglB (SODERGREN and KAISER 1983). Restriction analysis of single and double insertions at 01900 and 02224 is shown in Figure 2. The double insertion strains yield only two fragments that contain Tn5 sequences, showing that 01900 and 02224 lie between the same pair of adjacent EcoRI sites. These data along with the results of digestion with BamHI restriction endonuclease yield the map shown in Figure 3B. Four in- dependent estimates of the distance between Q1900 and Q2224 were computed from the map (Figure 3B, legend). Their average is 16.5 kb with a range of +1.

Genetic recombination between linked Tn5 insertions and comparison with the re- striction maps: To measure the recombination distance between two sites of T n 5 insertion, crosses were performed by transduction between a donor strain carrying T n 5 at one of the sites of insertion and a recipient strain carrying Tn5-132 at the other. Results of these Km’ Tcs X Km” Tc‘ transductions are presented in Table 2. Crosses 1, 2, 5 and 6 in Table 2 are important controls. For example, cross 1 01900 (Tn5-wt) X 01900 (Tn5-132) shows 100% cotrans- duction, confirming that 01900 (Tn5-wt) and Ql900 (Tn5-132) occupy the same locus on the Myxococcus chromosome and, therefore, behave as alleles in crosses. Crosses 2, 5 and 6 confirm that Tn5-wt and Tn5-132 behave as alleles at 02224, at 01933 and, with 3% exceptions, at 01902. The few ex- ceptions at 01902 are Km’ Tc‘ transductants, which could be transpositions, duplications or other rearrangements. The exceptions have not been investi- gated further.

Table 2 gives the cotransduction frequencies between Tn5s located at non- allelic sites, which measure the distance between those sites. Pairs of reciprocal crosses (cross 3 us. cross 4 and cross 7 us. cross 8) give the same frequency.

If it is assumed that DNA fragments of fixed size are cut at random from the Myxococcus chromosome by the generalized transducing phages Mx4 and Mx8, and that crossovers between the donor fragments and the recipient chro- mosome occur equally in all homologous regions, then the frequency of co- transduction, C, should vary with the fractional distance, t , separating the cotransduced markers according to C = [ l - t]’ (WU 1966). If WU’S calculation is made for transduction from a donor with Tn5 into a recipient without T n 5 at the homologous site, then the total length of donor DNA that is available for recombination would be the total amount of DNA packaged by the trans- ducing particle reduced by the length of Tn5. Because Mx4 packages 62 kb of DNA (GEISSELSODER, CAMPOS and ZUSMAN 1978 and Table 3, legend) and Mx8 packages 56 kb of DNA (MARTIN et al. 1978), the amount of DNA in a transducing particle that is available for recombination will be 50 kb (56-5.7) for Mx8 and 56 kb (62-5.7) for Mx4. The fractional distance t , then, becomes

Page 8: RECOMBINATION IN THE VICINITY OF INSERTIONS OF …DK3225 R2224(Tn5-wt)Ql900(Tn5-132) Mx4(DK2777) X DK1850 Each independent insertion of the transposon Tn5 is identified by an R number

288 E. SODERGREN E T AL.

TABLE 2

Cotransduction ~~ ~~~~~

Cotransduc- Donor, Tn5-wt Recipient, Tn5- 132 Km' transductants tion

Cross Site Strain Phage Site Strain Tc' Tc' %

1 01900 DK1900 Mx4 Q1900 DK1850 0 107 100 2 02224 DK2777 Mx4 Q2224 DK1870 0 219 100 3 01900 DK1900 Mx4 Q2224 DK1870 268 112 30 4 02224 DK2777 Mx4 Q1900 DK1850 125 58 31

5 01902 DK1902 Mx8 91902 DK1851 1 1 307 97 6 01933 DK1933 Mx8 01933 DK1853 0 120 100 7 01902 DK1902 Mx8 01933 DK1853 412 534 56 8 01933 DK1933 Mx8 01902 DK1851 241 299 54

Donor strains were resistant to kanamycin (Tn5-wt), and recipients were resistant to tetracycline (Tn5-132). Transduction mixtures were plated on kanamycin medium to select kanamycin-resistant transductants. Each kanamycin-resistant transductant was picked and tested for its response to oxytetracycline. Cotransduction is the percentage of transductants having the donor phenotype Km' Tc".

the ratio of the distance separating the cotransduced markers to the total distance that is available for recombination. Application of Wu's formula to the observed cotransduction frequencies yields map distances that agree with the distances measured by restriction fragment size for both intervals (Table 3).

DISCUSSION

Three pieces of data reported here support the proposition that transposon Tn5 is a well-behaved genetic marker in transduction experiments. In spite of structural differences between donor and recipient chromosomes arising from the presence of T n 5 in one but not the other, Tn5 recombines like a point mutation in most types of crosses in M. xanthus. First, Tn5 and Tn5-132 inserted at the same chromosomal locus behave as genetic alleles and trans- ductants inherit either one or the other (Table 2). Exceptions, which arise at a frequency of a few percent or less, could result from transposition in the donor or the recipient. Second, reciprocal two-factor crosses in which both markers are Tn5s show the same frequency of cotransduction (Table 2). De- spite the unequal contributions of DNA made by donor and recipient to a transduction, the equality of cotransduction frequencies in reciprocal transduc- tions indicates that the recombination process itself is symmetrical. Third, the agreement between the observed recombination frequencies and those pre- dicted by applying Wu's function to the physical distance between insertion sites (Table 3) implies that the recombination probability is proportional to the distance between sites.

Point 2, equality of reciprocal cotransduction frequencies, deserves addi- tional comment because equality is not always observed when only one of the two markers is a transposon. For example, with a series of short deletions in

Page 9: RECOMBINATION IN THE VICINITY OF INSERTIONS OF …DK3225 R2224(Tn5-wt)Ql900(Tn5-132) Mx4(DK2777) X DK1850 Each independent insertion of the transposon Tn5 is identified by an R number

RECOMBINATION NEAR T N ~ 289

TABLE 3

Comparison of cotransduction with restriction map

Cotransduction fre- Recombination dis- Mav interval Phage quencv. tanceb Phvsical distance‘

Q1900-Q2224 Mx4 30 -C 3% 18 zk 2 kb 16.5 f 0.5 kb Q1902-Ql933 Mx8 55 -e 9% 9 + 2 k b 10.8 f 1 kb

a Cotransduction frequencies are from Table 2. Distance calculated from Wu’s ( 1 966) function for 50 kb (phage Mx8) and 56 kb (phage Mx4)

of transduced DNA, where calculated distance = 50 ( 1 - C”) or 56 ( 1 - C”) and C is the cotransduction frequency. The total length of Mx4 DNA was calculated from the ratio of length of Mx4 DNA to PM2 DNA of 6.02 (GEISSELSODER, CAMPOS and ZUSMAN 1978), from the ratio of length of PM2 DNA to XDNA of 0.213 (KRIEGSTEIN and HOGNFSS 1974) and from the length of XDNA of 48.5 kb (SANGER et al. 1982).

Data from Figures 1 , 2 and 3 with average values and range taken as described in the text.

the hisG gene of Salmonella typhimurium, F. CHUMLEY and M. JOHNSTON found higher frequencies of cotransduction when T n 10 was the selected marker and inheritance of his’ was screened than when his’ was selected and TnlO was screened (KLECKNER, ROTH and BOTSTEIN 1977). This finding appears para- doxical because a common set of transducing DNA fragments, which carry both TnlO and his‘, ought to be responsible for cotransduction in both ex- periments. However, the cotransduction frequency is the ratio of the number of cotransductants to the total number of transductants for each marker taken singly. The paradox arises because the total number of TnlO transductants is smaller than the total number of his+ transductants. As pointed out by KLECK- NER, ROTH and BOTSTEIN (1977), a higher cotransduction frequency is found when TnlO is selected because fewer transductants are expected to contain the whole of TnlO than the whole of his+. (TnlO is 9.3 kb in length and the his deletions tested had removed less than 1 kb.) By contrast, in two-factor crosses with two Tn5 insertions, the same frequency of total transductants would be expected for each marker, yielding equal reciprocal cotransduction frequencies.

At least two limitations to the recombination behavior of transposons as point mutations can be found. First, recombinations can occur between homologous segments in Tn5 and Tn5-132 located at different sites, giving rise to tandem duplications of chromosomal segments (AVERY and KAISER 1983). However, fewer than 1% of Km‘ recombinants in crosses of the type described here are found to be duplications. Second, when two regions of structural heterozygos- ity are very close to each other, they may impair base pairing and recombi- nation in the intervening region. HOPPE et al. (1979) suggest that a failure to observe recombination between TnlO and a deletion less than about 50 base pairs away might be explained by impairment of base pairing.

Major differences in transduction frequency from marker to marker are observed in M. xanthus as in other organisms. For example, resistance to no- vobiocin was found to be transduced fourfold more frequently than cglF by the same stock of Mx8 (SODERGREN and KAISER 1983). These differences can be explained in at least three ways: differential replication of the donor chro-

Page 10: RECOMBINATION IN THE VICINITY OF INSERTIONS OF …DK3225 R2224(Tn5-wt)Ql900(Tn5-132) Mx4(DK2777) X DK1850 Each independent insertion of the transposon Tn5 is identified by an R number

290 E. SODERGREN ET AL.

mosome, differences in selection efficiency for different phenotypes or differ- ences in the distribution of sites in host DNA at which the packaging of DNA into transducing particles can start UACKSON, LASKI and ANDRES 1982). Which- ever explanation is appropriate, the agreement between physical and transduc- tional distances implies that Mx8 and Mx4 general transducing particles tend to package random fragments of the Myxococcus donor genome in the vicinity of each marker transduced.

This investigation was supported by National Institutes of Health research grant AG 2908 from the National Institute on Aging. E.S. was a fellow of the American Cancer Society and subsequently of the National Institute of Allergy and Infectious Disease.

We wish to thank RONALD GILL for discussion and encouragement; JOHN CARLSON, CARL MANN and STEWART SCHERER for purified restriction enzymes; and JERRY KUNER and GEORGE WEIN- STOCK for DNA samples.

LITERATURE CITED

AUERSWALD, E.-A., G. LUDWIG and H. SCHALLER, 1981

AVERY, L. and D. KAISER, 1983

BERG, D. E., 1977

Structural analysis of Tn5. Cold Spring Harbor Symp. Quant. Biol. 45: 107-113.

In situ transposon replacement and isolation of a spontaneous tandem genetic duplication in Myxococcus xanthus. Mol. Gen. Genet. In press.

Insertion and excision of the transposable kanamycin resistance determinant. pp. 205-212. In: DNA Insertion Elements, Plasmids, and Episomes, Edited by A. I. BUKHARI, J. A. SHAPIRO and S. L. ADHYA. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

BERG, D. E., J. DAVIES, B. ALLET and J.-D. ROCHAIX, 1975 Transposition of R factor genes to bacteriophage A. Proc. Natl. Acad. Sci. USA 72: 3628-2632.

BERG, D. E., C. EGNER, B. J. HIRSCHEL, J. HOWARD, L. JOHNSRUD, R. A. JORGENSEN and T. D. TLSTY, 1978 Insertion, excision, and inversion of Tn5. Cold Spring Harbor Symp. Quant. Biol. 45: 115-123.

Polarity of T n 5 insertion mutations in Esche- BERG, D. E., A. WEISS, and L. CROSSLAND, 1980 richia coli. J. Bacteriol. 142: 439-446.

CAMPBELL, A,, P. STARLINGER, D. E. BERG, D. BOTSTEIN, E. M. LEDERBERG, R. P. NOVICK and W. SZYBALSKI, 1979 Nomenclature of transposable elements in prokaryotes. Plasmid 2: 466- 473.

DAVIS, R. W., D. BOTSTEIN and J. R. ROTH, 1980 Advanced Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

GEISSELSODER, J., J. M. CAMPOS and D. R. ZUSMAN, 1978 Physical characterization of bacterio- phage Mx4, a generalized transducing phage for M~XOCOCCUS xanlhus. J. Mol. Biol. 1 1 9 179- 189.

HOPPE, I., H. M. JOHNSTON, D. BIEK and J. R. ROTH, 1979 A refined map of the hi& gene of Salmonella typhimurium. Genetics 92: 17-26.

JACKSON, E. N., F. LASKI and C. ANDRES, 1982 Bacteriophage P22 mutants that alter the speci- ficity of DNA packaging. J. Mol. Biol. 154: 551-563.

JORGENSEN, R. A., D. E. BERG, B. ALLET and W. S. REZNIKOFF, 1979 Restriction enzyme cleavage map of TnlO, a transposon which encodes tetracycline resistance. J. Bacteriol. 137: 681-685.

A restriction enzyme cleavage map of T n 5 and location of a region encoding neomycin resistance. Mol. Gen. Genet. 177:

JORGENSEN, R. A., S. J. ROTHSTEIN and W. S. REZNIKOFF, 1979

65-72.

Page 11: RECOMBINATION IN THE VICINITY OF INSERTIONS OF …DK3225 R2224(Tn5-wt)Ql900(Tn5-132) Mx4(DK2777) X DK1850 Each independent insertion of the transposon Tn5 is identified by an R number

RECOMBINATION NEAR T N ~ 291

KLECKNER, N., R. K. CHAN, B.-K. TYE and D. BOTSTEIN, 1975 Mutagenesis by insertion of a drug-resistance element carrying an inverted repetition. J. Mol. Biol. 97: 561-575.

KLECKNER, N., J. ROTH and D. BOTSTEIN, 1977 Genetic engineering in vivo using translocatable drug-resistance elements: new methods in bacterial genetics. J. Mol. Biol. 116 125-159.

KRIEGSTEIN, H. J. and D. S. HOCNESS, 1974 Mechanism of DNA replication in Drosophila chro- mosomes: structure of replication forks and evidence for bidirectionality. Proc. Natl. Acad. Sci. USA 71: 135-139.

Uses of transposon Tn5 in the genetic analysis of Myxococcus xanthus. In: Microbiology 198 1 , Edited by D. Schlessinger. American Society for Microbiology, Washington, D.C.

KUNER, J. M. and D. KAISER, 1981 lntroduction of transposon Tn5 into Myxococcus for analysis of developmental and other nonselectable mutants. Proc. Natl. Acad. Sci. USA 7 8 425-429.

MARTIN, S., E. SODERGREN, T. MASUDA and D. KAISER, 1978 Systematic isolation of transducing phages for Myxococcus xanthus. Virology 88: 44-53.

MCDONELL, M. W., M. N. SIMON and F. W. STUDIER, 1977 Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J. Mol. Biol. 110: 119-146.

Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113:

ROTHSTEIN, S. J., R. A. JORGENSEN, J. C.-P. YIN, Z. YONG-DI, R. C. JOHNSON and W. S. REZNIKOFF,

SANGER, F., A. R. COULSON, G. F. HONG, D. F. HILL and G. B. PETERSON, 1982 Nucleotide sequence of bacteriophage X DNA. J. Mol. Biol. 162: 729-773.

SODERGREN, E. and D. KAISER, 1983 Insertions of Tn5 near genes that govern stimulatable cell motility in Myxococcus. J. Mol. Biol. 167: 295-310.

SOUTHERN, E. M., 1975 Detection of specific sequences aniong DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-51 7.

WAHL, G, M., R. A. PADGETT and G. R. STARK, 1979 Gene amplification causes overproduction of the first three enzymes of UMP synthesis in N-(Phosphonacety1)-L-aspartate-resistant ham- ster cells. J. Biol. Chem. 254, 8679-8689.

WAHL, G. M., M. STERN and G. R. STARK, 1979 Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc. Natl. Acad. Sci. USA 76, 3683-3687.

Wu, T. T., 1966 A model for three-point analysis of random general transduction. Genetics 54:

Corresponding editor: D. BOTSTEIN

KUNER, J. M., L. AVERY, D. E. BERG and D. KAISER, 1981

RIGBY, P. W., M. DIECKMANN, C. RHODFS and P. BERG, 1977

237-251.

1981 Genetic organization of Tn5. Cold Spring Harbor Symp. Quant. Biol. 45: 99-105.

405-410.