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Reversal of mutator phage Mu integration
J. Mol. Biol. (1976) 96, 87-99
Reversal of Mutator Phage Mu Integration A. I. BTJKHARI Cold Spring Harbor Laboratory Cold Spring Harbor, N. Y. 11724, U.S.A. (Received 10 Decetiber 1974) The temperate bacteriophage Mu cause8 mutation8 by inserting its DNA randomly into the genes of its host bacterium Eachwichiu c&i. It is shown here that Mu DNA can be precisely excised from the different integration sites and that a~ a result wild-type function of the gene into which Mu was inserted is restored. The excision of Mu DNA is observable only if the Mu prophage carries mutations at the X locus. Thus, lee+ revertants from six strains, containing heat-inducible prophage Mu cta62 at different locations in the 2 gene of the lac operon, were readily obtained by flrat introducing the X mutation into Mu cts62. The lac+ revertants produced wild-type /l-galactosidase, and no trace of Mu DNA could be detected in them; this indicates that the junction of Mu DNA and host DNA can be specifically recognized. However, the excision of Mu DNA is generally not perfect, because in most cases it does not lead to the wild-type genotype. The function of gene A of Mu appears to be required for excision. Since the lethal functions of Mu are completely blocked in the Mu &a62 X prophage, the X locus probably has a regulatory function. At least one X mutation is caused by an insertion of about 900 base-pairs in Mu DNA. The discovery of the X mutants opens the way for studying the reversible interaction of the host and Mu chromosomes, and for using Mu to manipulate the host genome in various ways.
1. Introduction The mutator phage Mu causes mutations in its host bacterium Escherichia coli (Taylor, 1963). It has been established that mutations result from the stable, linear insertion of Mu DNA within the affected genes (Martuscelli et al., 1971) and that Mu DNA can be inserted at multiple sites within a single gene (Bukhari & Zipser, 1972 ; Daniel1 et al., 1972). Bukhari & Zipser (1972) concluded that Mu integrates its DNA into the host genome at random points, to establish lysogeny. Lysogenization of E. wli cells by Mu is perhaps the only known example of such highly promiscuous integration of one DNA molecule into another. Even the small insertions found to occur spontaneously in the E. coli chromosome cluster at hot spots (Starlinger t Saedler, 1972). Although no specific host sequence appears to be required, Mu uses a particular site on its own DNA for integration. As a result, all Mu prophages have the same gene order irrespective of their sites of insertion (Abelson et al., 1973). However, Mu can integrate in either of the two orientations with respect to host markers. Since the phage and prophage genomes are colinear, it is likely that Mu DNA ends are involved in the integration process (Hsu & Davidson, 1972; Wijffelman et aZ., 1973). Whether Mu integration is a reversible process, that is, whether Mu DNA can be excised from the different sites on the host chromosome, has remained an enigma. 87
88
A. I.
BUKHARI
Since Mu is inserted randomly, it follows that host sequences adjacent to the Mu prophages are different, and therefore the junction of Mu DNA and host DNA1 generates new sequences in each case. If Mu DNA can be excised precisely, the excision proteins must be capable of making specific cuts in the sequences, which differ from prophage to prophage. One evidence of the precise excision of Mu DNA would be the reversion of Mu-induced mutations. However, up until now the mutations caused by the insertion of Mu have been stable and have not been shown to revert at a detectable frequency, as originally reported by Taylor (1963). Jordan et al. (1968) described a rare case of reversion which occurred at a frequency of less than lo-la. The revertants that can normally be isolated in some cases from tho Mu-induced mutants have been shown to arise because of secondary site mutations and not because of the excision of Mu DNA (Bukhari & Taylor, 1971). In contrast, the bacteriophage X-induced mutations, which can be obtained by forcing /\ into abnormal integration sites, revert readily (Shimada et aE., 1972). Furthermore, h lysogens containing heat-inducible derivatives of X can be easily cured of their prophages by a short heat treatment, but Mu lysogens carrying heat-inducible Mu mutants cannot be cured of their prophages by a simple heat treatment. This paper reports the development of a system which allows the isolation of revertants from the Mu-induced mutants and the recovery of cells completely cured of Mu DNA. The excision of Mu DNA in this system can be precise, that is, the wildtype host sequences are regenerated, but is frequently imprecise, that is, the wild-type gene function is not restored. The discovery of the excision of Mu DNA stemmed from the isolation of the X mutants of Mu. The X mutants are obtained by plating Mu cts lysogens, containing thermosensitive Mu repressor, at 42”C, at which temperature the prophage is induced, killing the host cells. Some of the surviving cells contain the Mu X mutants which are defective prophages. The X mutations have pleiotropic effects, apparently eliminating both lethal and essential functions of the phage. Mu DNA is spontaneously excised from the Mu cts X lysogens, and cells completely free of Mu DNA can be readily recovered.
2. Materials and Methods (a) BacteriaE
and phage &?-a&s
The bacterial strains used, all derivatives of E. coli K12, are listed in Table 1. Phage Mu-l is referred to as Mu. The clear-plaque mutants of Mu (Mu c), the amber mutants (Mu am), and the temperature-sensitive mutants (Mu Is) have been described previously (Bukhari & Metlay, 1973; Bukhari & Curtin, 1974). The heat-inducible derivative of Mu, presumably containing a mutation in the repressor gene c, was Mu ets62 (Abelson et al., 1973; Howe, 1973). (b) Genetic nomenclature Symbols that denote genotypic and phenotypio traits have been defined earlier (Bukhnri & Metlay, 1973). The symbol : : is used to indicate that Mu is inserted in the gene preceding the symbol. For example, ZacZ8305 : :(Mu cts62) means that the heat-inducible Mu prophage is inserted into the 2 gene of the Eat operon. The number 8305 identifies the Mu insertion whose location within the 2 gene has been determined by deletion mapping.
(c) Genetic procedures A Tryptone/yeast extract medium (LB broth) was used for routine cultivation of bacteria as well as for dilution of phages. Minimal medium plates contained 1*6*/e agar in M9 salt8 supplemented with a carbon Bource at a final concentration of 0.2% and with required
REVERSAL
OF Mu INTEGRATION TABLE
1
List of bacterial strains Strain
40 FPLS014 BU8638 BU8304 BU8305 BUS306 BUS308 BU8364 BU8366 BUS366 BU8367 BUS368 BUS23 BU624
Relevant markers
sex
FF’ FF F
F’ F’ F F’
F’ F F’
F’ F’
Reference
Apro lae trp-8 St+ F’ pro + la0 +1Apro lao Apro lm his - me-t- SW Mun F/pro+ lacZ8304: : (Mucte62)/Apw kzc, hia-, met-, StrRMuR F’pro+ ZacZ8306: :(Mu ct&32)/Apro Zac,his-, met-, St+ Mun F’pro+ ZacZ8306: :(Mu cte62)/Apro lac, hie-, met-, StrR MuR F’pro+ ZacZ8308: : (Mu &62)/Apro Zac, his-, met-, StrR MuR F’pro+ &z&8364: :(Mu&62)/Apro luc, his-, met-, StrRMuR F’pro+ lucZ83bb: :(Mucts62)/Apro luc, his-, met-, StrRMuR F’pro+ lacZ8366: :(Muc&62)/Apwlac, his-, met-, StrRMuR F’pro+ ZucZ8367: : (Mu &362)/Apro lac, his-. met-, Stra MuR F’pro+ ZucZ8368: : (Mu &62)/Apro lac, hia-, met-, Stra Muk F’ pro+ ZacZ8306: : (Mu c&162XbOOO)/Apm lac Mu cts62 If’ pro + ZacZ8368: : (Mu c&62 XbOO4)/Apro la0 Mu u&62
MuR and Strk mean resistance to phage Mu and to streptomycin, indicated by A.
Bukhari t Metlay (1973) This work This work This work This work This work This work This work This work This work This work This work
respectively. Deletions are
amino acids at predetermined optimal concentrations varying from 20 to 40 pg/ml. M&or&y-lactose plates, on which lactose-fermenting (Lac + ) colonies become red, and XG plates (minimal medium containing &bromo-4-chloro-3-j-n-galactoside), on which Lac + colonies appear blue, were used to differentiate between Lac+ and Laccolonies. These media, the bacterial mating procedures, the preparation of phage lysates, and the assaying of the lysates by the agar overlay method have been described elsewhere (Bukhari t Metlay, 1973; Miller, 1972). To prepare Mu lysates by heat induction, the Mu ots62 lysogens were grown at 32°C shifted to 44°C for 30 min at a density of about 2 x 10s cells/ml, and the shaking was continued at 37°C. Liberation of phage by Mu lysogens was usually examined by replica plating of inocula from master plates on L-agar plates containing about lo* sensitive indicator bacteria embedded in a soft agar (0.7%) layer. Immunity to Mu was scored by spotting Mu o onto a streak of bacteria to be tested. To mutagenize bacteria with Mu ots62, the phage lysate was spotted onto the bacterial lawn and surviving lysogens from the spot were isolated after 12 h incubation at 32°C. (d) Biooherniml procedures Assay of fl-galactosidase and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to resolve /I-galactosidase polypeptides were as reported previously (Bukhari & Zipser, 1973). Mu particles were purified by cesium chloride density-gradient centrifugation after precipitation with polyethylene glycol and DNA wss extrrtcted with phenol (Bukhari & Allet, 1975). The DNAs were digested with the specific restriction endonucleases, and the resulting fragments were resolved by electrophoresis through 1.4Oh agarose gels in the presence of O-5 pg ethidium bromide/ml ss described by Sharp et aZ. (1973). The enzymes Hpa II from Hemophik ~ara;inJEwnzae (Sharp et al., 1973) and Eco*RI from E. ooZi RY 13 were gifts of Drs B. S. Zain and R. Roberts.
3. Results (a) Insertion
of MZL cts62 into the 1acZ gene
Since the lac operon has been dissected genetically in great detail, Mu insertions of the la& gene provided a well-characterized system to study the excision of Mu DNA.
A. I.
90
BUKHARI
Strain FPL5014, carrying an F’ pro + lac+ episome in a Apo luc background, was mutagenized with Mu cts62, and the Lac- mutants were isolated on XG plates. These Lac- strains had no p-galactosidase activity and had Mu cts62 inserted into the ZacZ gene on the F’ pro+ lac episome, as shown by the simultaneous transfer of the Mu prophage and the episome to the recipient bacteria in mating experiments. The insertions were mapped against a set of deletions removing different parts of the Z gene (Bukhari & Zipser, 1972). The orientation of the Mu genes, with respect to the lac operator, for each insertion was determined by the method of Zeldis et al. (1973) and, in some cases, by the prophage deletion method (Bukhari & Metlay, 1973). A map of the .?ucZ gene, giving the relative positions of the Mu cts62 insertions and their orientations, is shown in Figure 1. Z 8305
8308
8357
C
PO,
I 21
8354 cd5
flrannrm
wt/vswd
--
8304
I
---25?
8306
8358 Sk
II
l,y --
R’“-i---~=~4---27;j----1RI
Fxa. 1. A map of Mu cta62 insertions in the la& gene. The points of insertion are indicated by arrows. The numbers of insertions are shown above the jagged line representing Mu DNA. The 2 termini of Mu are marked by c (the immunity end) and S. The insertions were mapped as described by Bukhari & Zipser (1972). The reference ZacZ deletions are shown under the line. Two other insertions used, 8366 and 8366, gave no recombination with the deletions or point mutations of the Zuc genes, and are not shown in the map.
The F’ pro+ lacZ :: (Mu cts62) episomes were transferred to the Apro lac strain BU8538, which is Mu-resistant because of a lack of adsorption sites for Mu. This step was necessary to eliminate superinfection of cells in heat-induction experiments. (b) Survival of Mu cts62 lysogens at 42°C Mu Gts lysogens are killed at 42°C because of prophage induction. The strains containing the F’ pro + lacZ: : (Mu cts62) episomes in Mun background were plated at 42”C, to see whether cells cured of Mu prophages or Lac+ revertants with F’ pro+ TABLE
2
Survival of Mu cts26 lysogens at 42°C No. viable cells/ml at 32°C
No. viable cells/ml et 42%
Strain
Medium
BU8306
Minimal Nutrient
1.6 x 10s 1.9 x 100
2.9 x 104 1.1 x 106
BU8368
Minimal Nutrient
1.6 x 10s 1.7 x 10s
4.6 x lo* 2.2 x 108
The cultures were grown in LB broth at 32°C and plated at 32°C and 42°C on minimal supplemented with histidine and methionine, and on Tryptone plates.
medium,
REVERSAL
OF Mu
INTEGRATION
91
luc + episomea could be isolated. The survival frequencies of these strains are shown in Table 2. The number of surviving cells was higher on rich media, since the cells that had lost the episome and therefore had become pro- Mu- could grow on these media. The number of cells on the rich media thus retlected the frequency of the spontaneous loss of the F’ pro+ ZucZ: : (Mu cts62) episome. On minimal medium, only those cells that retained the epiaome could form colonies. None of the survivors on minimal medium was found to be Lac + . The survivors fell into three major classes: (1) deletion mutants, which had part or all of the Mu genome missing (Bukhari & Metlay, 1973; Howe, 1973). Deletions that removed all of the Mu DNA generally extended beyond the luc operon. (2) Suppressor-sensitive mutants, all of which were shown to contain mutations in one Mu gene (Bukhari & Howe, unpublished results). The gene apparently codes an early Mu function. (3) Mutants containing defective prophages, presumably because of non-suppressible mutations in Mu. Most of these defective prophagea were the X mutants as described in a later section. (c) The revertible clones Although all survivors of the heat treatment, from the ZucZ:: (Mu cts) strains, remained Lac -, colonies belonging to class 3 gave Lac + sectors after prolonged incubation on McConkey-lactose plates. The thermoresistant survivors from each strain were therefore systematically examined for their ability to revert to Lac+. As shown in Table 3, a number of survivors from six of the nine F’ pro+ lacZ: : (Mu cts62) strains studied gave Lac+ revertants. The Lac+ revertants were shown to contain F’ pro+ kc+ episomes by mating experiments, an indication that MU DNA had been precisely excised, regenerating the wild-type episome. AS discussed below, the Lac+ revertants had normal /3-galactosidase and had no detectable trace of MU TABLE
3
Isolation of revertible clones from F’ pro + IacZ: : (Mu cts62) strains str6in
No. of colonies isolated at 42°C
BUS304 BU8306 BU8306 BU8308 BU8364 BU8356 BU8366 BUS367 BU8368
600 1700 300 300 200 300 300 400 1000
No. giving Lac + rev&ants 0 162 10 7 4 0 0 11 144
The cultures were grown at 32°C in LB broth and plated on minimal medium plates, supplemented with histidine and methionine, at 42°C. The colonies obtained were screened for their ability to revert to Lac+ by replica plating on McConkey-lactose plates. The revert,ing colonies showed minute red colonies w-ithin the Lac- clones.
A. I.
92
BUKHARI
I
Phenotype
lac genotype
-- I,PO,
2
__ I, PO ,
z
Mu cts
z
,Y
z
,Y
--
Lac-
Mu cts
--
Lac-
Mu def
Lac+
Mu-
--
FICA 2. Reversion
of the Mu-induoed
mutationa
of the la&
gene through
an intermediate
step.
DNA left at the original site of insertion. This reversion of the Mu-induced ZacZ mutations through an intermediate step is shown diagrammatically in Figure 2. The threa strains that did not give any revertible clones were later shown to have deletions of various sizes at the point of insertion (Howe & Zipser, personal communication). The removal of Mu DNA in these cases thus would still leave a lesion in the lac operon, accounting for the absence of Lac+ revertants. (d) The n&we of prophage in the revertible clones The revertible clones carried lucZ mutations which behaved like original Mu insertions in deletion mapping. Mu immunity tests, done after transfer of the episomes to a Mu-sensitive background (strain 40), indicated that these strains contained thermosensitive Mu repressor, as is the case with Mu cte62 lysogens. However, these strains contained defective prophages, since the cells were not killed upon heat induction and plaque-forming Mu particles could not be recovered at normal fiequency (less than one plaque-forming particle/108 cells). To see whether the revertible clones contained intact Mu DNA, marker rescue tests were done against a set of 16 Mu amber mutants, by transferring the F’ pro+ lac episomes to Su- Mu anz lysogens as described by Bukhari & Metlay (1973). All 16 markers were rescued from the episome of each of the revertible clones, an indication that the original Mu insertion was largely undamaged. Final proof that Mu DNA was intact was obtained by characterizing the rare plaque-forming particles, which could be isolated from the cultures of the revertible clones at a frequency of 10e8 to 10e9 per cell. The particles, which presumably arose because of reversion of the prophage mutation, were completely indistinguishable from the parental Mu cts62 by both biological and physical criteria. The DNA molecules of these particles were cleaved with Hernophilus influemae endonucleases, and the fragments generated were compared with those of the Mu c&62 parental DNA (Bukhari t Allet, 1975). No differences could be detected. Therefore, the revertible clones from different strains had complete Mu DNA at the original sites of insertion. The mutations which caused inactivation of the prophage in the revertible clones are termed the X mutations. No Mu X amber mutants could be isolated, even after N-methyl-N’-nitrosoguanidine mutagenesis, an indication that the X mutations may not be simple point mutations. To examine the DNA of the X mutants directly, the Mu X prophages were rescued with a Mu ck62 helper phage. The F’ PO + .!acZ : : (Mu ($862 X) episomes were transferred to a Apro lac strain lysogenic for Mu cts62. The Mu cts62 X/Mu cte6i diploids (strains BU523 and BU624) were heat-induced, and the phage particles,
REVERSAL
OF Mu INTEGRATION
93
presumably a mixture of Mu c&62 X and Mu c&62, were purified by cesium chloride density centrifugation. The phenol-extracted DNA molecules from these phage preparations were cleaved with the specific endonuclease HpaIIt (Sharp et al., 1973), and the fragments obtained were separated electrophoretically on agarose gels. Plate I shows the gel patterns obtained from the two Mu cts62 X + Mu cts62 preparations along with the patterns obtained from the X revertants and the normal Mu DNA molecules. It can be seen that all readily identifiable bands are the same in Mu c, Mu cJs62, Mu c&62 X5000 preparations and the X revertants. However, the Mu cts62 X5004 DNA sample differs from Mu cts62 X5000 as well as other Mu DNA samples in that it shows a prominent new fragment, which is absent in the X5004 revertant examined. This indicates that Mu cts62 X5004 contains an insertion. That an insertion is responsible for the X5004 mutations was confirmed by the analysis shown in Plate II. EcosRI endonuclease produces a fragment of about 5100 basepairs from the c gene end of Mu DNA (Allet & Bukhari, 1975). The mixture of Mu cts62 X5004 and Mu cts62 DNA molecules yields two fragments, one migrating with the 5100 fragment and the other corresponding to an approximate size of 6000 base-pairs. It can be inferred that the Eco*RI fragment in the case of X5004 DNA contains an insertion of about 900 base-pairs. (e) The excision of phuge Mu DNA The F’ pro+ lacZ: : (Mu c&s62X) strains were plated on minimal medium plates containing either lactose or melibiose as a sole source of carbon, to measure the frequencies of the Lac + and melibiose + (Mel + ) revertants. Since growth on minimallactose plates requires ,%galactosidase activity, the number of L&c+ revertants would indicate the frequency of the precise excision of Mu DNA, restoring functional Z gene. On the other hand, the transport and hence utilization of the sugar melibiose at 41°C does not require Z gene function but requires a functional ZucY gene, encoding lactose permeate (see Miller, 1972). Since the insertions are completely polar (Bukhari t Zipser, 1972), growth on melibiose would require removal of Mu DNA, relieving polarity, whether or not the wild-type Z gene is restored. Thus, the number of Mel+ revertants would reveal both precise and imprecise excision of Mu DNA. As shown in Table 4, the frequency of Lac+ revertants was about IO-” per cell at 37°C the optimal temperature for scoring revertants. The number of revertants was not higher at 42°C. However, there appeared to be some inhibition of growth of Lac + revertants at 42°C and the reversion frequencies were not reproducible. Mel + revertants were at least tenfold more than L&c+ revertants. Furthermore, 100/b of the randomly picked Mel+ revertants were also Lac +. This indicated that although the excision of Mu DNA in Mu cts X lysogens is not generally perfect, it is highly specific: in about one out of ten detectable cases the cut is made exactly at the junction of Mu DNA and host DNA. The Lac+ revertants synthesized the same amount of fi-galactosidase, upon induction, as the Lac+ parent, and the enzyme could not be differentiated from the wild-type enzyme by heat-inactivation kinetics or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The revertants were also found to have lost Mu t Abbreviations used: Hps II, restriction endonuolease II of Hemophilua Eoo .RI, reatrictioti endonuole~e Rl of B. ~08 RY13 (RTF-1).
paminjluenzae;
A. I.
94
BUKHARI
TABLE 4 Frequency
of ,!A+
and Mel + revertants in F’ pro + 1acZ : : (iWu cts62 X) clo~res
Strain
No. of cells plated
h’o. of L&c+ colonies
pu’o. of Mel+ colonies
8306-x6000 -X6001 -X6002 8368-X6004 -X6006 -X6006 8306-X6013 8364-X6009 8367-X6012
10s 108 10s 108 108 10s 10s 10s 108
136 62 78 111 164 96 26 41 74
1400 1660 966 1600 1800 1460 410 1200 680
yh Mel + colon& also Lac + 7.7 8.5 8.3 2.6 10 7.6 7.8 7.6 8
The F’ pro+ ZucZ: :(Mu cts62 X) episomes from the revertible clones, of the different strains shown by number, were transferred to the Mu-sensitive trp- strain 40, and the derived strains were scored for Lao + and Mel + revertants. For Lac + revertants, the cells were plated on minimal the cells were plated on minimal lactose + tryptophan plates at 37°C and for Mel+ revertants melibiose + tryptophan plates at 41%. The Mel+ colonies were replica plated on McConkeylactose plates for identification of those which were also Lao + .
immunity and did not show rescue of any Mu marker tested. By all criteria, thus, the process of Mu integration had been precisely reversed in these cases. Rarely, Lac+ revertants with lower p-galactosidase activity than the wild-type strain were isolated. The nature of these revertants is being investigated. The Mel+ revertants that remained Lac- because of lesions in the Z gene had also lost Mu DNA completely, aa shown by the loss of Mu immunity and lack of any detectable Mu marker. A large number of the Mel+ revertants were screened for the synthesis of inactive p-galactosidase polypeptides by sodium dodecyl sulfatepolyacrylamide gel electrophoresis of the crude cell extracts. The polypeptide patterns obtained from 17 independent Mel+ revertants, originating from three different Mu insertions, are shown in Plate III. In three of these cases, new polypeptides can be /UC2: : (Mu cts) lo-‘- 10-e I /UC2: : (Mu cts Xl 10-S
10-S
A l0c.Z: Mu-
IocZL Mu; Mel+
10-s ii No reversion
Fm. 3. Various
events involved
in the reversion
lOCZ j
Mu-
of the la&:
: (Mu cts62) mutations.
I Mu crs
2 MUX -kMu
3 MUX +MU
4 X rev
5 X re”
6 X rev
7 Muc
PLATE 1 I. Analysis of Mu DNA molecules by the restriction ondonucleaso Eco~ HJ. The DNA samples were digested with Eco.KI as outlined in Plate 1. The sample at left labelrtl Mu X + Mu is the DNA of Mu particles from strain BU524 (Mu cts62 X5004/Mu cts62 diploid), and the sample labeled Mu is the DNA of Mu cts62 particles from strain BU8305. Mu DNA gives one isolated fragment of about 5100 base-pairs, whereas a mixture of Mu and Mu X DNAs yields two fragments, one of 5100 base-pairs and t,he other of about 6000 base-pairs.
[‘LATE 111. Analysis g1.1 eiect,rophorcGs.
of cell extract,s
of Mel + rr:vert,ants
by sodium
tiotl~gl
sulfatr-pr)lyacrylar~~i(ll~
The Mel+ revertants, obtained from the ZacZ: : (Mu cts62 X) strains, and other control cuiturrts v WR grown in 5 ml of LB broth in the presence of 5 ,? 10 -4 &l-isopropyl thio-8.u-galactopyran[)sitlr (II gratuitou-i inducer of the Zac operon), unle+ otherwisp indicated. The cells were centrifuged. rliirupted by heating in sodium dodecyl sulfate sample buffer, and analyzed by electrophoresis in a slab-type polyacrylemide gel (10%) as described earlier (Bukhari 8: Zipser, 1973). After rlnctrf,. phorosis the gels were stained with 0.25% Coomassie brilliant blue (in 5?/0 methanol, 7.59:, acetic acid). The samples are as follows: no. 24 is the wild-type Znc+ strain FPL5014 grown without isopropyl thio-/l-n-galaotopyranoside (UI), and no. 25 is the wild-t,ype strain grown wit,h intlucf>r (I ). An arrow shows the wild-type /Sgelactosidase monomer which appears in the induced culture*. NW I and 2 are the uninduced and induced cultures of BID305 (ZncZ8305: :(Mu rt.962)) and 3. 4 art’ uninduced and induced cultures of BU8358. No identifiable /I-galact,osidase polypept,icle 1* No. 5 is a Lar a~rtn in the induced samples. All samples from 5 t)o 22 were grown with inducer. rt,x-c>rtant from ZrtcZ8305 : : (Mu cts62 X6000) insertion, showing wild-type 8.galeotosidase rnonom~~~‘. Sample 6 t)o IO are Mel + revertants from ZacZ8305: : (Mu cts62 X6000) insertion; II to I i ar’~ .Zlel + r’evert,ants from ZacZ8358: : (Mu c&62 X5004) insertion; and 18 to 22 are MeI+ from Lw% 8354: :(Mu cts62 X5009) insertion. Three new polypeptides produced by t)he revcrtantq a~ indicated by arrows. No. 11 shows an inactive /3-galact,osidase polypeptide which is indi&nguish aide from the active monomer of 135,000 mol. wt,, whereas nos 7 and 19 xhow polyprydirlc hwvinp nlol. lvts brtween 110,000 and 120,000.
REVERSAL
OF Mu
96
INTEGRATION
seen. Revertant 7 from insertion 8305 and revertant 19 from insertion 8354 produce polypeptides distinctly smaller than the wild-type /3-galactosidase monomer, whereas revertant 11 from insertion 8358 synthesizes an inactive fl-galactosidase protein which is indistinguishable from the active monomer on gels. The presence of polypeptides smaller than the wild-type gene product, at least in two cases, indicates that the excision of Mu DNA can result in the deletion of some of the adjacent host DNA. Some of the other Mel+ revertants, which did not show deletions either by genetic recombination tests or by polypeptide analysis, were found to revert further to Lac + at a low frequency. These second-stage L&c+ revertants could be detected upon prolonged incubation on McConkey-lactose plates (frequency ~10~g/cell). This result suggests the presence of small insertions in the Z gene, left behind after Mu excision, which can be further removed to give the wild-type kc+ operon. A flow sheet of the events involved in the excision of Mu DNA is summarized in Figure 3. (f) Involvement of phage Mu functions To test the effect of wild-type Mu and different mutants of Mu on the excision of Mu DNA, the F’ pro+ lucZ: : (Mu cts62 X) episomes were transferred to the F- Mu lysogenio strains. As shown in Table 5A, the presence of a secondary Mu c+ or a Mu cts62 prophage abolished the appearance of both Lac+ and Mel+ revertants. The 16 Mu amber mutants tested had the same effect. All of these am mutants complemented the X mutants, as shown by the release of the Mu am particles by the Mu diploid TABLE
5
Effect of Mu mutations on the excision of Mu DNA A.
F’ pro+ F’pro+ F/pro+ F’ pro+ F’ pro+ F’pro+
B.
F’pro+ F’pro+ F/pro+ F/pro+ F’ PO+ F‘ pro+
Genotype
lacZ8306: ZacZ8306: ZacZ8306: ZacZ8368: EocZ8368: ZtccZ8306:
Survival at 42°C No. viable cells at 42°C No. viable cells at 32°C
:(Mu ctc62 XbOOO)/Apro lac, Mu:(Mucta62)/Apro Zac, Muc+ : (Mu c&62 X6OOO)/Apro lac, Mu cl8627 :(Mu cb62 X6004)/Apro Zac, Mu: (Mu cti62 X4006)/Ap+o Zac, Mu c+ : (Mu &a62 X6OOO)/Apro lac, Mu c+ amI
Genotype
ZacZ8306: : (Mu ct862 Ats6074) ZncZ8306: :(Mucts62 46134) ZacZ8306: : (Mu cts62 Bts6061) ZacZ8306: :(Mu cts62 Buam6176)$ la&8306 : : (Mu cts62 Pam6136) ZacZ8306: : (Mu Cl862 Ram6024)
-1 f-1
Frequency of revertants Lac + Mel + -10-s < lo-‘0 < 10-10 -10-s
-10-s
O/crevertible clones among survivors at 42’C < 0.06 < 0.06 8 6 11 14
t :Lac* revertants were scored at 42°C. In all other cases Lae+ revertants were scored at 37°C and Mel+ at 41°C. f Mu amber mutants were 16 different Mu mutants in Su- background (Bukhari & Metlay, 1973), as follows: Eam6002, Eam3021, Bam3007, #‘amBOll, Karn6008, Lam6026, Mam3022, Oam6023, Qa4n3018, Qam6001, Rum3008, Sam6014, am6002, anz6006, am6018, am6024. 3 Bu mutants do not complement known B gene mutants.
96
A. I. BUKHARI
cells. Thus, the X mutations are recessive. The inhibitory effect of secondary Xc prophages on the appearance of revertants may reflect the killing of the host cells after excision. To determine which Mu gene is responsible for the excision of Mu DNA, lucZ: : (Mu cts62) lysogens containing mutations in various Mu genes were plated at 42°C and the survivors were screened for the presence of revertible clones. Table 5B shows the effect of mutations in genes A, B, R, and F of Mu on the appearance of revertible clones. Genes A and B are early Mu genes, as the A and B mutants are unable to replicate their DNA and lyse the cells after induction; whereas, R and F are representatives of the late genes (Bukhari $ Curtin, 1974; Wijffelman et al., 1974). All strains except those carrying mutations in gene A gave revertible clones. Since lesions in gene A appear to result in a specific block in the excision of Mu DNA, the gene A product either is the Mu excision protein or controls the expression of the excision function.
4. Discussion It has been clearly demonstrated that Mu-induced mutations, caused by the insertion of Mu DNA within the affected genes, can revert. The reversion is the result of excision of Mu DNA accompanied by sealing of the host chromosome. A two-step process is required to observe this reversion, which is not normally detected. The first step is the isolation of revertible clones that contain defective prophages owing to X mutations. The Mu X prophages are unable to express their lytic functions and cannot kill the host cells upon induction. The second step involves selection of cells in which Mu DNA has been excised from the Mu X lysogens. Perhaps reversion of the Mu-induced mutations is not seen generally because of the lethal functions of the wild-type prophages, which kill the host cells after excision. Mu insertions of the ZucZ gene, located at different sites and with different orientations, are excised readily via the Mu X pathway, often resulting in conversion of the +. This restoration of the wild-type Z gene, without any trace Zac- genotype to l47.c of Mu DNA, is the final proof of the finding that integration of Mu can occur without causing any deletion at the point of Mu insertion (Bukhari & Zipser, 1972). In a few anomalous cases, however, no Lao+ clones could be recovered. These strains were subsequently found to contain relatively large deletions, covering most of the luc operon, which presumably arose during the process of Mu integration (Howe & Zipser, personal communication). We have shown that reversion of Mu-induced mutations also occurs in other genes, for example, in the arabinose operon (Razzaki t Bukhari, unpublished results). Reznikoff has observed the reversion of trp : : (Mu) mutants to trp + (personal communication). The excision of Mu DNA in the lacZ: : (Mu cts X) strains is perfect only in about one out of ten detectable cases. This is shown by the observations that Mel+ revertants are tenfold higher than Lac+ revertants and that 10% of the randomly picked Mel+ revertants are simultaneously Lao + . Since reversion to Mel + requires only the removal of Mu DNA, resulting in the release of polarity, and not the restoration of the wild-type Z gene, it seems that in most cases a part of the adjacent host DNA is excised with Mu DNA. In some cases, however, the Mel+ revertants can further revert to Lac + . It is possible that in these cases a small non-polar insertion is retained at the Mu integration site.
REVERSAL
OF Mu
97
INTEGRATION
The excision .of Mu’DNA~in the Mu cts X lysogens is Mwpcifio, 88 iti is blouked by a secondary prophage in tram and by mutations in gene A in cis. The function of gene A is particularly interesting, since it apparently controls the excision of Mu DNA. Results not presented here also show that the recA mutations, which affect the recombination system of the host cells (Clark, 1973), cause a reduction in the number of Lac + revertants from the lacZ : : (Mu cts X) strains. It is not clear whether the recA function acts in conjunction with Mu-excision functions. It is possible that the recA function is involved in the repair of the host chromosome after excision. Any model of Mu integration and excision must take into account the unusual features of Mu DNA. The S gene end of vegetative Mu DNA (mol. wt 25 x 106) is variable in length (Daniell et al., 1973). The variable region amounts to about 3% of the total length and has sequence which differ from molecule to molecule. The c gene end also appears to have a small amount of variation in length (Bukhari & Allet, 1975). The perfect excision of Mu DNA suggests that the large variable region of Mu, with random sequences, is not present in the prophage state, since it would be diffioult to imagine a mechanism by which the junction of this variable region with the host DNA could be specifically recognized. The absence of the variable region in three independent prophages has been demonstrated by electron microscope heteroduplex studies (Hsu & Davidson, 1974). However, it is possible that a very small part of the S variable end is integrated with the phage DNA during the lysogenization process, and this accounts for the presumed insertions left behind after Mu excision. Alternatively, it can be proposed that the sequences at the Mu prophage ends are duplicated and that a part of the duplicated region can be left behind after excision. In either case, in order for excision to be Mu-specific, the excision proteins must recognize the Mu ends and cut the DNA within a narrow range, the cut being sometimes precise but mostly imprecise. The X mutants of Mu are interesting because they are the only Mu mutants known in which killing functions of Mu seem to be totally eliminated. The X locus probably has a regulatory function controlling the expression of various Mu genes. Wijffelman & van de Putte (personal communication) have found that some of the early Mu transcription and most of the late transcription is blocked in the X mutants. However, the excision of Mu DNA indicates that some functions continue to be expressed, albeit at a low rate, in the X mutants, The X mutations revert at a low frequency, giving rise to normal plaque-forming Mu particles. The DNA molecules of these
A
A’
C
Mu I
8’
c
swy
I I
A
A
A
I A’ 8
8’
FIG. 4. Fusion of 2 host DNA segments using Mu. The host sequences are represented by AA’, WY, and BB’. Mu genomes are shown by the jagged lines, c and 8 being the two Mu termini.
98
A. I.
BUKHARI
revertant particles are indistinguishable from the wild-type Mu DNA, as shown by restriction endonuclease analysis. Thus, the X mutations are not deletions. However, at least one X mutation is caused by an insertion of about 900 base-pairs. Probably most X mutations are insertions of some kind, since the X mutations revert with a frequency of about 10-s per cell and X am mutants cannot be isolated. The excision of Mu DNA via the Mu X pathway can be exploited to manipulate the host genome in various ways. A simple case of gene fusion is illustrated in Figure 4. The WY region in the Figure is deleted because of the host-promoted recombination between the two Mu prophages, leaving the sequences AA’ separated from BB’ by a single prophage. Excision of this prophage via the Mu X pathway would result in the fusion of AA’ with BB’. This principle can be applied to transpose and to alter different segments of the host genome. The excision of Mu DNA described here is reminiscent of the excision of spontaneous insertions, termed insertosomes, found in the genome of E. co.5 (Malamy et al., 1972; Starlinger & Saedler, 1972). The transposition of the control elements in maize also appears to involve the precise removal of DNA segments (McClintock, 1961; Brink $ Williams, 1973). The mechanisms of excision in all such cases might have elements in common. I am indebted to David Zipser for continuous discussions and support, and to B. Sayeeda Zain for gifts of restriction endonucleases and for helping me with the analysis of DNA samples. I am also thankful to M. Howe for refining the mapping of some of the Mu insertions, and to D. Botstein for critically reviewing the manuscript. This work was supported by grants to the author from the National Science Foundation (GB-43280) and the Jane Coffin Childw Memorial Fund for Medical Research (no. 298), and also by a grant to D. Zipser from the Institute of General Medical Sciences, National Institutes of Health. REFERENCES Abelson, J., Boram, W., Bukhari, A. I., Faelen, M., Howe, M., Metlay, M., Taylor, A. L., Toussaint, A., van de Putte, P., Westmaas, G. C. & Wijffelman, C. A. (1973). ‘Virology, 54, 9&92. Allet, B. & Bukhari, A. I. (1975). J. MOE. Biol. 92, 529-540. Brink, R. A. & Williams, E. (1973). Cenetice, 73, 273-296. Bukhari, A. I. & Allet, B. (1975). T/‘iroZogy, 63, 30-39. Bukhari, A. I. & Curtin, P. (1974). J. Viral. 14, 1615-1616. Bukhari, A. I. & Metlay, M. (1973). ViFoZogy, 54, 109-116. Bukhari, A. I. & Taylor, A. L. (1971). J. Bactetiol. 105, 844-854. Bukhari, A. I. & Zipser, D. (1972). Nature New BioZ. 236, 240-243. Bukhari, A. I. & Zipser, D. (1973). Nature New BioZ. 243, 238-241. Clark, A. J. (1973). Annu. Rev. Genet. 7, 67-86. Daniell, E., Roberts, R. & Abelson, J. (1972). J. Mol. BioZ. 69, 1-8. Daniell, E., Boram, W. & Abelson, J. (1973). Ppoc. Not. Ad. Sci., U.S.A. 70, 2163-2156. Howe, M. (1973). Virology, 54, 93-101. Hsu, M. & Davidson, N. (1972). Proc. Nat. Aad. Sci. U.S.A., 69, 2823-2827. Hsu, M. & Davidson, N. (1974). ViTology, 58, 229-239. Jordan, E., Saedler, H. & Starlinger, P. (1968). Mol. Gen. Genet. 102, 363-363. Melamy, M. H., Fiandt, M. & Szybalski, W. (1972). MOE. Gen. Gemt. 119, 207-222. Martuscelli, J., Taylor, A. L., Cummings, D., Chapman, V., DeLong, S. BE Caiieda, L, (1971). J. V&oZ..8, 661-663. McClintock, B. (1961). Amer. Natural. 95, 265-277.
REVERSAL
OF Mu INTEGRATION
99
Miller, J. H. (1972). Experiments ilt Mo2t~u..%~ GenetiM, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Sharp, P., Sugden, B. & Sambrook, J. (1973). Btichemktry, 12, 3055-3063. Shimada, K., Weisberg, R. A. t Gottesman, M. E. (1972). J. MOE. Bio.!. 63, 483-503. Starlinger, P. & Saedler, H. (1972). Biochimie, 54, 177-185. Taylor, A. L. (1963). Proc. Nat. AC&. Sci., U.S.A. 50, 1043-1051. Wijffelman, C. A., Westmaas, G. C. & van de Putte, P. (1973). %“iroZogy, 54, 125-134. Wijffelman, C. A., Gas&r, M., Stevens, W. I?. & van de Putto, P. (1974). Mol. Gen.
CJenet.131,85-96. Zeldis, J., Bukhari,
A. I. & Zipser, D. (1973). ViroZogy, 55, 289-294.
Reversal of Mutator Phage Mu Integration A. I. BTJKHARI Cold Spring Harbor Laboratory Cold Spring Harbor, N. Y. 11724, U.S.A. (Received 10 Decetiber 1974) The temperate bacteriophage Mu cause8 mutation8 by inserting its DNA randomly into the genes of its host bacterium Eachwichiu c&i. It is shown here that Mu DNA can be precisely excised from the different integration sites and that a~ a result wild-type function of the gene into which Mu was inserted is restored. The excision of Mu DNA is observable only if the Mu prophage carries mutations at the X locus. Thus, lee+ revertants from six strains, containing heat-inducible prophage Mu cta62 at different locations in the 2 gene of the lac operon, were readily obtained by flrat introducing the X mutation into Mu cts62. The lac+ revertants produced wild-type /l-galactosidase, and no trace of Mu DNA could be detected in them; this indicates that the junction of Mu DNA and host DNA can be specifically recognized. However, the excision of Mu DNA is generally not perfect, because in most cases it does not lead to the wild-type genotype. The function of gene A of Mu appears to be required for excision. Since the lethal functions of Mu are completely blocked in the Mu &a62 X prophage, the X locus probably has a regulatory function. At least one X mutation is caused by an insertion of about 900 base-pairs in Mu DNA. The discovery of the X mutants opens the way for studying the reversible interaction of the host and Mu chromosomes, and for using Mu to manipulate the host genome in various ways.
1. Introduction The mutator phage Mu causes mutations in its host bacterium Escherichia coli (Taylor, 1963). It has been established that mutations result from the stable, linear insertion of Mu DNA within the affected genes (Martuscelli et al., 1971) and that Mu DNA can be inserted at multiple sites within a single gene (Bukhari & Zipser, 1972 ; Daniel1 et al., 1972). Bukhari & Zipser (1972) concluded that Mu integrates its DNA into the host genome at random points, to establish lysogeny. Lysogenization of E. wli cells by Mu is perhaps the only known example of such highly promiscuous integration of one DNA molecule into another. Even the small insertions found to occur spontaneously in the E. coli chromosome cluster at hot spots (Starlinger t Saedler, 1972). Although no specific host sequence appears to be required, Mu uses a particular site on its own DNA for integration. As a result, all Mu prophages have the same gene order irrespective of their sites of insertion (Abelson et al., 1973). However, Mu can integrate in either of the two orientations with respect to host markers. Since the phage and prophage genomes are colinear, it is likely that Mu DNA ends are involved in the integration process (Hsu & Davidson, 1972; Wijffelman et aZ., 1973). Whether Mu integration is a reversible process, that is, whether Mu DNA can be excised from the different sites on the host chromosome, has remained an enigma. 87
88
A. I.
BUKHARI
Since Mu is inserted randomly, it follows that host sequences adjacent to the Mu prophages are different, and therefore the junction of Mu DNA and host DNA1 generates new sequences in each case. If Mu DNA can be excised precisely, the excision proteins must be capable of making specific cuts in the sequences, which differ from prophage to prophage. One evidence of the precise excision of Mu DNA would be the reversion of Mu-induced mutations. However, up until now the mutations caused by the insertion of Mu have been stable and have not been shown to revert at a detectable frequency, as originally reported by Taylor (1963). Jordan et al. (1968) described a rare case of reversion which occurred at a frequency of less than lo-la. The revertants that can normally be isolated in some cases from tho Mu-induced mutants have been shown to arise because of secondary site mutations and not because of the excision of Mu DNA (Bukhari & Taylor, 1971). In contrast, the bacteriophage X-induced mutations, which can be obtained by forcing /\ into abnormal integration sites, revert readily (Shimada et aE., 1972). Furthermore, h lysogens containing heat-inducible derivatives of X can be easily cured of their prophages by a short heat treatment, but Mu lysogens carrying heat-inducible Mu mutants cannot be cured of their prophages by a simple heat treatment. This paper reports the development of a system which allows the isolation of revertants from the Mu-induced mutants and the recovery of cells completely cured of Mu DNA. The excision of Mu DNA in this system can be precise, that is, the wildtype host sequences are regenerated, but is frequently imprecise, that is, the wild-type gene function is not restored. The discovery of the excision of Mu DNA stemmed from the isolation of the X mutants of Mu. The X mutants are obtained by plating Mu cts lysogens, containing thermosensitive Mu repressor, at 42”C, at which temperature the prophage is induced, killing the host cells. Some of the surviving cells contain the Mu X mutants which are defective prophages. The X mutations have pleiotropic effects, apparently eliminating both lethal and essential functions of the phage. Mu DNA is spontaneously excised from the Mu cts X lysogens, and cells completely free of Mu DNA can be readily recovered.
2. Materials and Methods (a) BacteriaE
and phage &?-a&s
The bacterial strains used, all derivatives of E. coli K12, are listed in Table 1. Phage Mu-l is referred to as Mu. The clear-plaque mutants of Mu (Mu c), the amber mutants (Mu am), and the temperature-sensitive mutants (Mu Is) have been described previously (Bukhari & Metlay, 1973; Bukhari & Curtin, 1974). The heat-inducible derivative of Mu, presumably containing a mutation in the repressor gene c, was Mu ets62 (Abelson et al., 1973; Howe, 1973). (b) Genetic nomenclature Symbols that denote genotypic and phenotypio traits have been defined earlier (Bukhnri & Metlay, 1973). The symbol : : is used to indicate that Mu is inserted in the gene preceding the symbol. For example, ZacZ8305 : :(Mu cts62) means that the heat-inducible Mu prophage is inserted into the 2 gene of the Eat operon. The number 8305 identifies the Mu insertion whose location within the 2 gene has been determined by deletion mapping.
(c) Genetic procedures A Tryptone/yeast extract medium (LB broth) was used for routine cultivation of bacteria as well as for dilution of phages. Minimal medium plates contained 1*6*/e agar in M9 salt8 supplemented with a carbon Bource at a final concentration of 0.2% and with required
REVERSAL
OF Mu INTEGRATION TABLE
1
List of bacterial strains Strain
40 FPLS014 BU8638 BU8304 BU8305 BUS306 BUS308 BU8364 BU8366 BUS366 BU8367 BUS368 BUS23 BU624
Relevant markers
sex
FF’ FF F
F’ F’ F F’
F’ F F’
F’ F’
Reference
Apro lae trp-8 St+ F’ pro + la0 +1Apro lao Apro lm his - me-t- SW Mun F/pro+ lacZ8304: : (Mucte62)/Apw kzc, hia-, met-, StrRMuR F’pro+ ZacZ8306: :(Mu ct&32)/Apro Zac,his-, met-, St+ Mun F’pro+ ZacZ8306: :(Mu cte62)/Apro lac, hie-, met-, StrR MuR F’pro+ ZacZ8308: : (Mu &62)/Apro Zac, his-, met-, StrR MuR F’pro+ &z&8364: :(Mu&62)/Apro luc, his-, met-, StrRMuR F’pro+ lucZ83bb: :(Mucts62)/Apro luc, his-, met-, StrRMuR F’pro+ lacZ8366: :(Muc&62)/Apwlac, his-, met-, StrRMuR F’pro+ ZucZ8367: : (Mu &362)/Apro lac, his-. met-, Stra MuR F’pro+ ZucZ8368: : (Mu &62)/Apro lac, hia-, met-, Stra Muk F’ pro+ ZacZ8306: : (Mu c&162XbOOO)/Apm lac Mu cts62 If’ pro + ZacZ8368: : (Mu c&62 XbOO4)/Apro la0 Mu u&62
MuR and Strk mean resistance to phage Mu and to streptomycin, indicated by A.
Bukhari t Metlay (1973) This work This work This work This work This work This work This work This work This work This work This work
respectively. Deletions are
amino acids at predetermined optimal concentrations varying from 20 to 40 pg/ml. M&or&y-lactose plates, on which lactose-fermenting (Lac + ) colonies become red, and XG plates (minimal medium containing &bromo-4-chloro-3-j-n-galactoside), on which Lac + colonies appear blue, were used to differentiate between Lac+ and Laccolonies. These media, the bacterial mating procedures, the preparation of phage lysates, and the assaying of the lysates by the agar overlay method have been described elsewhere (Bukhari t Metlay, 1973; Miller, 1972). To prepare Mu lysates by heat induction, the Mu ots62 lysogens were grown at 32°C shifted to 44°C for 30 min at a density of about 2 x 10s cells/ml, and the shaking was continued at 37°C. Liberation of phage by Mu lysogens was usually examined by replica plating of inocula from master plates on L-agar plates containing about lo* sensitive indicator bacteria embedded in a soft agar (0.7%) layer. Immunity to Mu was scored by spotting Mu o onto a streak of bacteria to be tested. To mutagenize bacteria with Mu ots62, the phage lysate was spotted onto the bacterial lawn and surviving lysogens from the spot were isolated after 12 h incubation at 32°C. (d) Biooherniml procedures Assay of fl-galactosidase and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to resolve /I-galactosidase polypeptides were as reported previously (Bukhari & Zipser, 1973). Mu particles were purified by cesium chloride density-gradient centrifugation after precipitation with polyethylene glycol and DNA wss extrrtcted with phenol (Bukhari & Allet, 1975). The DNAs were digested with the specific restriction endonucleases, and the resulting fragments were resolved by electrophoresis through 1.4Oh agarose gels in the presence of O-5 pg ethidium bromide/ml ss described by Sharp et aZ. (1973). The enzymes Hpa II from Hemophik ~ara;inJEwnzae (Sharp et al., 1973) and Eco*RI from E. ooZi RY 13 were gifts of Drs B. S. Zain and R. Roberts.
3. Results (a) Insertion
of MZL cts62 into the 1acZ gene
Since the lac operon has been dissected genetically in great detail, Mu insertions of the la& gene provided a well-characterized system to study the excision of Mu DNA.
A. I.
90
BUKHARI
Strain FPL5014, carrying an F’ pro + lac+ episome in a Apo luc background, was mutagenized with Mu cts62, and the Lac- mutants were isolated on XG plates. These Lac- strains had no p-galactosidase activity and had Mu cts62 inserted into the ZacZ gene on the F’ pro+ lac episome, as shown by the simultaneous transfer of the Mu prophage and the episome to the recipient bacteria in mating experiments. The insertions were mapped against a set of deletions removing different parts of the Z gene (Bukhari & Zipser, 1972). The orientation of the Mu genes, with respect to the lac operator, for each insertion was determined by the method of Zeldis et al. (1973) and, in some cases, by the prophage deletion method (Bukhari & Metlay, 1973). A map of the .?ucZ gene, giving the relative positions of the Mu cts62 insertions and their orientations, is shown in Figure 1. Z 8305
8308
8357
C
PO,
I 21
8354 cd5
flrannrm
wt/vswd
--
8304
I
---25?
8306
8358 Sk
II
l,y --
R’“-i---~=~4---27;j----1RI
Fxa. 1. A map of Mu cta62 insertions in the la& gene. The points of insertion are indicated by arrows. The numbers of insertions are shown above the jagged line representing Mu DNA. The 2 termini of Mu are marked by c (the immunity end) and S. The insertions were mapped as described by Bukhari & Zipser (1972). The reference ZacZ deletions are shown under the line. Two other insertions used, 8366 and 8366, gave no recombination with the deletions or point mutations of the Zuc genes, and are not shown in the map.
The F’ pro+ lacZ :: (Mu cts62) episomes were transferred to the Apro lac strain BU8538, which is Mu-resistant because of a lack of adsorption sites for Mu. This step was necessary to eliminate superinfection of cells in heat-induction experiments. (b) Survival of Mu cts62 lysogens at 42°C Mu Gts lysogens are killed at 42°C because of prophage induction. The strains containing the F’ pro + lacZ: : (Mu cts62) episomes in Mun background were plated at 42”C, to see whether cells cured of Mu prophages or Lac+ revertants with F’ pro+ TABLE
2
Survival of Mu cts26 lysogens at 42°C No. viable cells/ml at 32°C
No. viable cells/ml et 42%
Strain
Medium
BU8306
Minimal Nutrient
1.6 x 10s 1.9 x 100
2.9 x 104 1.1 x 106
BU8368
Minimal Nutrient
1.6 x 10s 1.7 x 10s
4.6 x lo* 2.2 x 108
The cultures were grown in LB broth at 32°C and plated at 32°C and 42°C on minimal supplemented with histidine and methionine, and on Tryptone plates.
medium,
REVERSAL
OF Mu
INTEGRATION
91
luc + episomea could be isolated. The survival frequencies of these strains are shown in Table 2. The number of surviving cells was higher on rich media, since the cells that had lost the episome and therefore had become pro- Mu- could grow on these media. The number of cells on the rich media thus retlected the frequency of the spontaneous loss of the F’ pro+ ZucZ: : (Mu cts62) episome. On minimal medium, only those cells that retained the epiaome could form colonies. None of the survivors on minimal medium was found to be Lac + . The survivors fell into three major classes: (1) deletion mutants, which had part or all of the Mu genome missing (Bukhari & Metlay, 1973; Howe, 1973). Deletions that removed all of the Mu DNA generally extended beyond the luc operon. (2) Suppressor-sensitive mutants, all of which were shown to contain mutations in one Mu gene (Bukhari & Howe, unpublished results). The gene apparently codes an early Mu function. (3) Mutants containing defective prophages, presumably because of non-suppressible mutations in Mu. Most of these defective prophagea were the X mutants as described in a later section. (c) The revertible clones Although all survivors of the heat treatment, from the ZucZ:: (Mu cts) strains, remained Lac -, colonies belonging to class 3 gave Lac + sectors after prolonged incubation on McConkey-lactose plates. The thermoresistant survivors from each strain were therefore systematically examined for their ability to revert to Lac+. As shown in Table 3, a number of survivors from six of the nine F’ pro+ lacZ: : (Mu cts62) strains studied gave Lac+ revertants. The Lac+ revertants were shown to contain F’ pro+ kc+ episomes by mating experiments, an indication that MU DNA had been precisely excised, regenerating the wild-type episome. AS discussed below, the Lac+ revertants had normal /3-galactosidase and had no detectable trace of MU TABLE
3
Isolation of revertible clones from F’ pro + IacZ: : (Mu cts62) strains str6in
No. of colonies isolated at 42°C
BUS304 BU8306 BU8306 BU8308 BU8364 BU8356 BU8366 BUS367 BU8368
600 1700 300 300 200 300 300 400 1000
No. giving Lac + rev&ants 0 162 10 7 4 0 0 11 144
The cultures were grown at 32°C in LB broth and plated on minimal medium plates, supplemented with histidine and methionine, at 42°C. The colonies obtained were screened for their ability to revert to Lac+ by replica plating on McConkey-lactose plates. The revert,ing colonies showed minute red colonies w-ithin the Lac- clones.
A. I.
92
BUKHARI
I
Phenotype
lac genotype
-- I,PO,
2
__ I, PO ,
z
Mu cts
z
,Y
z
,Y
--
Lac-
Mu cts
--
Lac-
Mu def
Lac+
Mu-
--
FICA 2. Reversion
of the Mu-induoed
mutationa
of the la&
gene through
an intermediate
step.
DNA left at the original site of insertion. This reversion of the Mu-induced ZacZ mutations through an intermediate step is shown diagrammatically in Figure 2. The threa strains that did not give any revertible clones were later shown to have deletions of various sizes at the point of insertion (Howe & Zipser, personal communication). The removal of Mu DNA in these cases thus would still leave a lesion in the lac operon, accounting for the absence of Lac+ revertants. (d) The n&we of prophage in the revertible clones The revertible clones carried lucZ mutations which behaved like original Mu insertions in deletion mapping. Mu immunity tests, done after transfer of the episomes to a Mu-sensitive background (strain 40), indicated that these strains contained thermosensitive Mu repressor, as is the case with Mu cte62 lysogens. However, these strains contained defective prophages, since the cells were not killed upon heat induction and plaque-forming Mu particles could not be recovered at normal fiequency (less than one plaque-forming particle/108 cells). To see whether the revertible clones contained intact Mu DNA, marker rescue tests were done against a set of 16 Mu amber mutants, by transferring the F’ pro+ lac episomes to Su- Mu anz lysogens as described by Bukhari & Metlay (1973). All 16 markers were rescued from the episome of each of the revertible clones, an indication that the original Mu insertion was largely undamaged. Final proof that Mu DNA was intact was obtained by characterizing the rare plaque-forming particles, which could be isolated from the cultures of the revertible clones at a frequency of 10e8 to 10e9 per cell. The particles, which presumably arose because of reversion of the prophage mutation, were completely indistinguishable from the parental Mu cts62 by both biological and physical criteria. The DNA molecules of these particles were cleaved with Hernophilus influemae endonucleases, and the fragments generated were compared with those of the Mu c&62 parental DNA (Bukhari t Allet, 1975). No differences could be detected. Therefore, the revertible clones from different strains had complete Mu DNA at the original sites of insertion. The mutations which caused inactivation of the prophage in the revertible clones are termed the X mutations. No Mu X amber mutants could be isolated, even after N-methyl-N’-nitrosoguanidine mutagenesis, an indication that the X mutations may not be simple point mutations. To examine the DNA of the X mutants directly, the Mu X prophages were rescued with a Mu ck62 helper phage. The F’ PO + .!acZ : : (Mu ($862 X) episomes were transferred to a Apro lac strain lysogenic for Mu cts62. The Mu cts62 X/Mu cte6i diploids (strains BU523 and BU624) were heat-induced, and the phage particles,
REVERSAL
OF Mu INTEGRATION
93
presumably a mixture of Mu c&62 X and Mu c&62, were purified by cesium chloride density centrifugation. The phenol-extracted DNA molecules from these phage preparations were cleaved with the specific endonuclease HpaIIt (Sharp et al., 1973), and the fragments obtained were separated electrophoretically on agarose gels. Plate I shows the gel patterns obtained from the two Mu cts62 X + Mu cts62 preparations along with the patterns obtained from the X revertants and the normal Mu DNA molecules. It can be seen that all readily identifiable bands are the same in Mu c, Mu cJs62, Mu c&62 X5000 preparations and the X revertants. However, the Mu cts62 X5004 DNA sample differs from Mu cts62 X5000 as well as other Mu DNA samples in that it shows a prominent new fragment, which is absent in the X5004 revertant examined. This indicates that Mu cts62 X5004 contains an insertion. That an insertion is responsible for the X5004 mutations was confirmed by the analysis shown in Plate II. EcosRI endonuclease produces a fragment of about 5100 basepairs from the c gene end of Mu DNA (Allet & Bukhari, 1975). The mixture of Mu cts62 X5004 and Mu cts62 DNA molecules yields two fragments, one migrating with the 5100 fragment and the other corresponding to an approximate size of 6000 base-pairs. It can be inferred that the Eco*RI fragment in the case of X5004 DNA contains an insertion of about 900 base-pairs. (e) The excision of phuge Mu DNA The F’ pro+ lacZ: : (Mu c&s62X) strains were plated on minimal medium plates containing either lactose or melibiose as a sole source of carbon, to measure the frequencies of the Lac + and melibiose + (Mel + ) revertants. Since growth on minimallactose plates requires ,%galactosidase activity, the number of L&c+ revertants would indicate the frequency of the precise excision of Mu DNA, restoring functional Z gene. On the other hand, the transport and hence utilization of the sugar melibiose at 41°C does not require Z gene function but requires a functional ZucY gene, encoding lactose permeate (see Miller, 1972). Since the insertions are completely polar (Bukhari t Zipser, 1972), growth on melibiose would require removal of Mu DNA, relieving polarity, whether or not the wild-type Z gene is restored. Thus, the number of Mel+ revertants would reveal both precise and imprecise excision of Mu DNA. As shown in Table 4, the frequency of Lac+ revertants was about IO-” per cell at 37°C the optimal temperature for scoring revertants. The number of revertants was not higher at 42°C. However, there appeared to be some inhibition of growth of Lac + revertants at 42°C and the reversion frequencies were not reproducible. Mel + revertants were at least tenfold more than L&c+ revertants. Furthermore, 100/b of the randomly picked Mel+ revertants were also Lac +. This indicated that although the excision of Mu DNA in Mu cts X lysogens is not generally perfect, it is highly specific: in about one out of ten detectable cases the cut is made exactly at the junction of Mu DNA and host DNA. The Lac+ revertants synthesized the same amount of fi-galactosidase, upon induction, as the Lac+ parent, and the enzyme could not be differentiated from the wild-type enzyme by heat-inactivation kinetics or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The revertants were also found to have lost Mu t Abbreviations used: Hps II, restriction endonuolease II of Hemophilua Eoo .RI, reatrictioti endonuole~e Rl of B. ~08 RY13 (RTF-1).
paminjluenzae;
A. I.
94
BUKHARI
TABLE 4 Frequency
of ,!A+
and Mel + revertants in F’ pro + 1acZ : : (iWu cts62 X) clo~res
Strain
No. of cells plated
h’o. of L&c+ colonies
pu’o. of Mel+ colonies
8306-x6000 -X6001 -X6002 8368-X6004 -X6006 -X6006 8306-X6013 8364-X6009 8367-X6012
10s 108 10s 108 108 10s 10s 10s 108
136 62 78 111 164 96 26 41 74
1400 1660 966 1600 1800 1460 410 1200 680
yh Mel + colon& also Lac + 7.7 8.5 8.3 2.6 10 7.6 7.8 7.6 8
The F’ pro+ ZucZ: :(Mu cts62 X) episomes from the revertible clones, of the different strains shown by number, were transferred to the Mu-sensitive trp- strain 40, and the derived strains were scored for Lao + and Mel + revertants. For Lac + revertants, the cells were plated on minimal the cells were plated on minimal lactose + tryptophan plates at 37°C and for Mel+ revertants melibiose + tryptophan plates at 41%. The Mel+ colonies were replica plated on McConkeylactose plates for identification of those which were also Lao + .
immunity and did not show rescue of any Mu marker tested. By all criteria, thus, the process of Mu integration had been precisely reversed in these cases. Rarely, Lac+ revertants with lower p-galactosidase activity than the wild-type strain were isolated. The nature of these revertants is being investigated. The Mel+ revertants that remained Lac- because of lesions in the Z gene had also lost Mu DNA completely, aa shown by the loss of Mu immunity and lack of any detectable Mu marker. A large number of the Mel+ revertants were screened for the synthesis of inactive p-galactosidase polypeptides by sodium dodecyl sulfatepolyacrylamide gel electrophoresis of the crude cell extracts. The polypeptide patterns obtained from 17 independent Mel+ revertants, originating from three different Mu insertions, are shown in Plate III. In three of these cases, new polypeptides can be /UC2: : (Mu cts) lo-‘- 10-e I /UC2: : (Mu cts Xl 10-S
10-S
A l0c.Z: Mu-
IocZL Mu; Mel+
10-s ii No reversion
Fm. 3. Various
events involved
in the reversion
lOCZ j
Mu-
of the la&:
: (Mu cts62) mutations.
I Mu crs
2 MUX -kMu
3 MUX +MU
4 X rev
5 X re”
6 X rev
7 Muc
PLATE 1 I. Analysis of Mu DNA molecules by the restriction ondonucleaso Eco~ HJ. The DNA samples were digested with Eco.KI as outlined in Plate 1. The sample at left labelrtl Mu X + Mu is the DNA of Mu particles from strain BU524 (Mu cts62 X5004/Mu cts62 diploid), and the sample labeled Mu is the DNA of Mu cts62 particles from strain BU8305. Mu DNA gives one isolated fragment of about 5100 base-pairs, whereas a mixture of Mu and Mu X DNAs yields two fragments, one of 5100 base-pairs and t,he other of about 6000 base-pairs.
[‘LATE 111. Analysis g1.1 eiect,rophorcGs.
of cell extract,s
of Mel + rr:vert,ants
by sodium
tiotl~gl
sulfatr-pr)lyacrylar~~i(ll~
The Mel+ revertants, obtained from the ZacZ: : (Mu cts62 X) strains, and other control cuiturrts v WR grown in 5 ml of LB broth in the presence of 5 ,? 10 -4 &l-isopropyl thio-8.u-galactopyran[)sitlr (II gratuitou-i inducer of the Zac operon), unle+ otherwisp indicated. The cells were centrifuged. rliirupted by heating in sodium dodecyl sulfate sample buffer, and analyzed by electrophoresis in a slab-type polyacrylemide gel (10%) as described earlier (Bukhari 8: Zipser, 1973). After rlnctrf,. phorosis the gels were stained with 0.25% Coomassie brilliant blue (in 5?/0 methanol, 7.59:, acetic acid). The samples are as follows: no. 24 is the wild-type Znc+ strain FPL5014 grown without isopropyl thio-/l-n-galaotopyranoside (UI), and no. 25 is the wild-t,ype strain grown wit,h intlucf>r (I ). An arrow shows the wild-type /Sgelactosidase monomer which appears in the induced culture*. NW I and 2 are the uninduced and induced cultures of BID305 (ZncZ8305: :(Mu rt.962)) and 3. 4 art’ uninduced and induced cultures of BU8358. No identifiable /I-galact,osidase polypept,icle 1* No. 5 is a Lar a~rtn in the induced samples. All samples from 5 t)o 22 were grown with inducer. rt,x-c>rtant from ZrtcZ8305 : : (Mu cts62 X6000) insertion, showing wild-type 8.galeotosidase rnonom~~~‘. Sample 6 t)o IO are Mel + revertants from ZacZ8305: : (Mu cts62 X6000) insertion; II to I i ar’~ .Zlel + r’evert,ants from ZacZ8358: : (Mu c&62 X5004) insertion; and 18 to 22 are MeI+ from Lw% 8354: :(Mu cts62 X5009) insertion. Three new polypeptides produced by t)he revcrtantq a~ indicated by arrows. No. 11 shows an inactive /3-galact,osidase polypeptide which is indi&nguish aide from the active monomer of 135,000 mol. wt,, whereas nos 7 and 19 xhow polyprydirlc hwvinp nlol. lvts brtween 110,000 and 120,000.
REVERSAL
OF Mu
96
INTEGRATION
seen. Revertant 7 from insertion 8305 and revertant 19 from insertion 8354 produce polypeptides distinctly smaller than the wild-type /3-galactosidase monomer, whereas revertant 11 from insertion 8358 synthesizes an inactive fl-galactosidase protein which is indistinguishable from the active monomer on gels. The presence of polypeptides smaller than the wild-type gene product, at least in two cases, indicates that the excision of Mu DNA can result in the deletion of some of the adjacent host DNA. Some of the other Mel+ revertants, which did not show deletions either by genetic recombination tests or by polypeptide analysis, were found to revert further to Lac + at a low frequency. These second-stage L&c+ revertants could be detected upon prolonged incubation on McConkey-lactose plates (frequency ~10~g/cell). This result suggests the presence of small insertions in the Z gene, left behind after Mu excision, which can be further removed to give the wild-type kc+ operon. A flow sheet of the events involved in the excision of Mu DNA is summarized in Figure 3. (f) Involvement of phage Mu functions To test the effect of wild-type Mu and different mutants of Mu on the excision of Mu DNA, the F’ pro+ lucZ: : (Mu cts62 X) episomes were transferred to the F- Mu lysogenio strains. As shown in Table 5A, the presence of a secondary Mu c+ or a Mu cts62 prophage abolished the appearance of both Lac+ and Mel+ revertants. The 16 Mu amber mutants tested had the same effect. All of these am mutants complemented the X mutants, as shown by the release of the Mu am particles by the Mu diploid TABLE
5
Effect of Mu mutations on the excision of Mu DNA A.
F’ pro+ F’pro+ F/pro+ F’ pro+ F’ pro+ F’pro+
B.
F’pro+ F’pro+ F/pro+ F/pro+ F’ PO+ F‘ pro+
Genotype
lacZ8306: ZacZ8306: ZacZ8306: ZacZ8368: EocZ8368: ZtccZ8306:
Survival at 42°C No. viable cells at 42°C No. viable cells at 32°C
:(Mu ctc62 XbOOO)/Apro lac, Mu:(Mucta62)/Apro Zac, Muc+ : (Mu c&62 X6OOO)/Apro lac, Mu cl8627 :(Mu cb62 X6004)/Apro Zac, Mu: (Mu cti62 X4006)/Ap+o Zac, Mu c+ : (Mu &a62 X6OOO)/Apro lac, Mu c+ amI
Genotype
ZacZ8306: : (Mu ct862 Ats6074) ZncZ8306: :(Mucts62 46134) ZacZ8306: : (Mu cts62 Bts6061) ZacZ8306: :(Mu cts62 Buam6176)$ la&8306 : : (Mu cts62 Pam6136) ZacZ8306: : (Mu Cl862 Ram6024)
-1 f-1
Frequency of revertants Lac + Mel + -10-s < lo-‘0 < 10-10 -10-s
-10-s
O/crevertible clones among survivors at 42’C < 0.06 < 0.06 8 6 11 14
t :Lac* revertants were scored at 42°C. In all other cases Lae+ revertants were scored at 37°C and Mel+ at 41°C. f Mu amber mutants were 16 different Mu mutants in Su- background (Bukhari & Metlay, 1973), as follows: Eam6002, Eam3021, Bam3007, #‘amBOll, Karn6008, Lam6026, Mam3022, Oam6023, Qa4n3018, Qam6001, Rum3008, Sam6014, am6002, anz6006, am6018, am6024. 3 Bu mutants do not complement known B gene mutants.
96
A. I. BUKHARI
cells. Thus, the X mutations are recessive. The inhibitory effect of secondary Xc prophages on the appearance of revertants may reflect the killing of the host cells after excision. To determine which Mu gene is responsible for the excision of Mu DNA, lucZ: : (Mu cts62) lysogens containing mutations in various Mu genes were plated at 42°C and the survivors were screened for the presence of revertible clones. Table 5B shows the effect of mutations in genes A, B, R, and F of Mu on the appearance of revertible clones. Genes A and B are early Mu genes, as the A and B mutants are unable to replicate their DNA and lyse the cells after induction; whereas, R and F are representatives of the late genes (Bukhari $ Curtin, 1974; Wijffelman et al., 1974). All strains except those carrying mutations in gene A gave revertible clones. Since lesions in gene A appear to result in a specific block in the excision of Mu DNA, the gene A product either is the Mu excision protein or controls the expression of the excision function.
4. Discussion It has been clearly demonstrated that Mu-induced mutations, caused by the insertion of Mu DNA within the affected genes, can revert. The reversion is the result of excision of Mu DNA accompanied by sealing of the host chromosome. A two-step process is required to observe this reversion, which is not normally detected. The first step is the isolation of revertible clones that contain defective prophages owing to X mutations. The Mu X prophages are unable to express their lytic functions and cannot kill the host cells upon induction. The second step involves selection of cells in which Mu DNA has been excised from the Mu X lysogens. Perhaps reversion of the Mu-induced mutations is not seen generally because of the lethal functions of the wild-type prophages, which kill the host cells after excision. Mu insertions of the ZucZ gene, located at different sites and with different orientations, are excised readily via the Mu X pathway, often resulting in conversion of the +. This restoration of the wild-type Z gene, without any trace Zac- genotype to l47.c of Mu DNA, is the final proof of the finding that integration of Mu can occur without causing any deletion at the point of Mu insertion (Bukhari & Zipser, 1972). In a few anomalous cases, however, no Lao+ clones could be recovered. These strains were subsequently found to contain relatively large deletions, covering most of the luc operon, which presumably arose during the process of Mu integration (Howe & Zipser, personal communication). We have shown that reversion of Mu-induced mutations also occurs in other genes, for example, in the arabinose operon (Razzaki t Bukhari, unpublished results). Reznikoff has observed the reversion of trp : : (Mu) mutants to trp + (personal communication). The excision of Mu DNA in the lacZ: : (Mu cts X) strains is perfect only in about one out of ten detectable cases. This is shown by the observations that Mel+ revertants are tenfold higher than Lac+ revertants and that 10% of the randomly picked Mel+ revertants are simultaneously Lao + . Since reversion to Mel + requires only the removal of Mu DNA, resulting in the release of polarity, and not the restoration of the wild-type Z gene, it seems that in most cases a part of the adjacent host DNA is excised with Mu DNA. In some cases, however, the Mel+ revertants can further revert to Lac + . It is possible that in these cases a small non-polar insertion is retained at the Mu integration site.
REVERSAL
OF Mu
97
INTEGRATION
The excision .of Mu’DNA~in the Mu cts X lysogens is Mwpcifio, 88 iti is blouked by a secondary prophage in tram and by mutations in gene A in cis. The function of gene A is particularly interesting, since it apparently controls the excision of Mu DNA. Results not presented here also show that the recA mutations, which affect the recombination system of the host cells (Clark, 1973), cause a reduction in the number of Lac + revertants from the lacZ : : (Mu cts X) strains. It is not clear whether the recA function acts in conjunction with Mu-excision functions. It is possible that the recA function is involved in the repair of the host chromosome after excision. Any model of Mu integration and excision must take into account the unusual features of Mu DNA. The S gene end of vegetative Mu DNA (mol. wt 25 x 106) is variable in length (Daniell et al., 1973). The variable region amounts to about 3% of the total length and has sequence which differ from molecule to molecule. The c gene end also appears to have a small amount of variation in length (Bukhari & Allet, 1975). The perfect excision of Mu DNA suggests that the large variable region of Mu, with random sequences, is not present in the prophage state, since it would be diffioult to imagine a mechanism by which the junction of this variable region with the host DNA could be specifically recognized. The absence of the variable region in three independent prophages has been demonstrated by electron microscope heteroduplex studies (Hsu & Davidson, 1974). However, it is possible that a very small part of the S variable end is integrated with the phage DNA during the lysogenization process, and this accounts for the presumed insertions left behind after Mu excision. Alternatively, it can be proposed that the sequences at the Mu prophage ends are duplicated and that a part of the duplicated region can be left behind after excision. In either case, in order for excision to be Mu-specific, the excision proteins must recognize the Mu ends and cut the DNA within a narrow range, the cut being sometimes precise but mostly imprecise. The X mutants of Mu are interesting because they are the only Mu mutants known in which killing functions of Mu seem to be totally eliminated. The X locus probably has a regulatory function controlling the expression of various Mu genes. Wijffelman & van de Putte (personal communication) have found that some of the early Mu transcription and most of the late transcription is blocked in the X mutants. However, the excision of Mu DNA indicates that some functions continue to be expressed, albeit at a low rate, in the X mutants, The X mutations revert at a low frequency, giving rise to normal plaque-forming Mu particles. The DNA molecules of these
A
A’
C
Mu I
8’
c
swy
I I
A
A
A
I A’ 8
8’
FIG. 4. Fusion of 2 host DNA segments using Mu. The host sequences are represented by AA’, WY, and BB’. Mu genomes are shown by the jagged lines, c and 8 being the two Mu termini.
98
A. I.
BUKHARI
revertant particles are indistinguishable from the wild-type Mu DNA, as shown by restriction endonuclease analysis. Thus, the X mutations are not deletions. However, at least one X mutation is caused by an insertion of about 900 base-pairs. Probably most X mutations are insertions of some kind, since the X mutations revert with a frequency of about 10-s per cell and X am mutants cannot be isolated. The excision of Mu DNA via the Mu X pathway can be exploited to manipulate the host genome in various ways. A simple case of gene fusion is illustrated in Figure 4. The WY region in the Figure is deleted because of the host-promoted recombination between the two Mu prophages, leaving the sequences AA’ separated from BB’ by a single prophage. Excision of this prophage via the Mu X pathway would result in the fusion of AA’ with BB’. This principle can be applied to transpose and to alter different segments of the host genome. The excision of Mu DNA described here is reminiscent of the excision of spontaneous insertions, termed insertosomes, found in the genome of E. co.5 (Malamy et al., 1972; Starlinger & Saedler, 1972). The transposition of the control elements in maize also appears to involve the precise removal of DNA segments (McClintock, 1961; Brink $ Williams, 1973). The mechanisms of excision in all such cases might have elements in common. I am indebted to David Zipser for continuous discussions and support, and to B. Sayeeda Zain for gifts of restriction endonucleases and for helping me with the analysis of DNA samples. I am also thankful to M. Howe for refining the mapping of some of the Mu insertions, and to D. Botstein for critically reviewing the manuscript. This work was supported by grants to the author from the National Science Foundation (GB-43280) and the Jane Coffin Childw Memorial Fund for Medical Research (no. 298), and also by a grant to D. Zipser from the Institute of General Medical Sciences, National Institutes of Health. REFERENCES Abelson, J., Boram, W., Bukhari, A. I., Faelen, M., Howe, M., Metlay, M., Taylor, A. L., Toussaint, A., van de Putte, P., Westmaas, G. C. & Wijffelman, C. A. (1973). ‘Virology, 54, 9&92. Allet, B. & Bukhari, A. I. (1975). J. MOE. Biol. 92, 529-540. Brink, R. A. & Williams, E. (1973). Cenetice, 73, 273-296. Bukhari, A. I. & Allet, B. (1975). T/‘iroZogy, 63, 30-39. Bukhari, A. I. & Curtin, P. (1974). J. Viral. 14, 1615-1616. Bukhari, A. I. & Metlay, M. (1973). ViFoZogy, 54, 109-116. Bukhari, A. I. & Taylor, A. L. (1971). J. Bactetiol. 105, 844-854. Bukhari, A. I. & Zipser, D. (1972). Nature New BioZ. 236, 240-243. Bukhari, A. I. & Zipser, D. (1973). Nature New BioZ. 243, 238-241. Clark, A. J. (1973). Annu. Rev. Genet. 7, 67-86. Daniell, E., Roberts, R. & Abelson, J. (1972). J. Mol. BioZ. 69, 1-8. Daniell, E., Boram, W. & Abelson, J. (1973). Ppoc. Not. Ad. Sci., U.S.A. 70, 2163-2156. Howe, M. (1973). Virology, 54, 93-101. Hsu, M. & Davidson, N. (1972). Proc. Nat. Aad. Sci. U.S.A., 69, 2823-2827. Hsu, M. & Davidson, N. (1974). ViTology, 58, 229-239. Jordan, E., Saedler, H. & Starlinger, P. (1968). Mol. Gen. Genet. 102, 363-363. Melamy, M. H., Fiandt, M. & Szybalski, W. (1972). MOE. Gen. Gemt. 119, 207-222. Martuscelli, J., Taylor, A. L., Cummings, D., Chapman, V., DeLong, S. BE Caiieda, L, (1971). J. V&oZ..8, 661-663. McClintock, B. (1961). Amer. Natural. 95, 265-277.
REVERSAL
OF Mu INTEGRATION
99
Miller, J. H. (1972). Experiments ilt Mo2t~u..%~ GenetiM, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Sharp, P., Sugden, B. & Sambrook, J. (1973). Btichemktry, 12, 3055-3063. Shimada, K., Weisberg, R. A. t Gottesman, M. E. (1972). J. MOE. Bio.!. 63, 483-503. Starlinger, P. & Saedler, H. (1972). Biochimie, 54, 177-185. Taylor, A. L. (1963). Proc. Nat. AC&. Sci., U.S.A. 50, 1043-1051. Wijffelman, C. A., Westmaas, G. C. & van de Putte, P. (1973). %“iroZogy, 54, 125-134. Wijffelman, C. A., Gas&r, M., Stevens, W. I?. & van de Putto, P. (1974). Mol. Gen.
CJenet.131,85-96. Zeldis, J., Bukhari,
A. I. & Zipser, D. (1973). ViroZogy, 55, 289-294.