nature of deletions formed in response to is2 in a revertant of the gal3 insertion of e. coli

Molec. gen. Gent. 163, 189-196 (1978) © by Springer-Verlag 1978

Nature of Deletions Formed in Response to IS2 in a Revertant of the gal3 Insertion of E. coil

Asad Ahmed and Douglas Scraba

Departments of Genetics and Biochemistry. University of Alberta, Edmonton, Alberta T6G 2E9, Canada

Summary. The gal3 mutation of E. coli, which arose by the insertion of IS2 in the OP region of the gal operon, reverts spontaneously by excision of the IS2 to produce inducible revertants or by mutational al- terations of IS2 to produce constitutive revertants. However, ga13(2) strains bearing chlD-pgl deletions produce constitutive revertants alone. We proposed that deletions formed in the presence of IS2 terminate specifically at its right end, so that revertants arising by excision of IS2 fuse the gal genes to other promoters. Therefore, the revertants are exclusively constitutive.

The above hypothesis was tested by electron microscopy of IS2-specific deletions. Spontaneous chlD- pgl deletions were isolated from gaF331 (a revertant of gal3 which retains IS2) and transferred to 2gaI genomes. Electron microscopy of DNA heteroduplexes from these phages confirmed that all of the deletions examined have one end-point fixed at the right end of IS2, whereas their other end-points are variable. In each case, the complete IS2 element was apparently retained. This specificity was also detect- able in a revertant (gaF200) which retains only the right 1/5 portion of the IS2. The frequencies of these deletions were generally increased in constitutive revertants of gal3. Since a galO ~ mutant did not show a similar increase, it seems that this effect depends upon a base sequence provided by IS2. Moreover, the presence of prophage 2 contributes to the specificity and, in some instances, the frequency of IS2- specific deletions.

A mechanism for the formation of the IS2-specific deletions has been proposed. A base sequence located at, or near, the right end of IS2 is recognized and nicked by a specific endonuclease. The nick is enlarged by unidirectional, exonucleolytic degradation to produce deletions extending outwards from the

For offprints contact." Asad Ahmed

insertion. In constitutive revertants, the nicking site may be exposed to endonucleolytic attack more fre- quently.

Introduction

The gal3 mutation (Lederberg, 1960) was caused by the insertion of a 1.I 1.2kb I D N A sequence, identified as IS2, in the operator-promoter (OP) region of the gal operon of E. coli (reviewed by Ahmed, 1977). This finding provides satisfactory explanation for the extreme polarity and lack of response to cer- tain chemical mutagens exhibited by gal3. This mutation revert spontaneously to produce three kinds of gal ÷ revertants, viz., stable inducible, stable constitutive, and unstable constitutive (Hill and Echols, 1966; Morse, 1967). We have shown that the stable inducible revertants arise by accurate excision of the IS2, whereas the stable constitutive revertants arise by partial deletions of IS2 which remove the rho-sensitive transcription termination site. The unstable revertants have been believed, in the past, to arise by the inversion of IS2 or by tandem duplications of the gal operon. In a separate communication (Ahmed and Johansen, 1978), we have suggested that these rever- rants arise by double (or multiple) mutations which inactivate the rho site and thus allow constitutive transcription. We have proposed that an IS2 element in a state of transcription acts as a hot spot for DNA repair, and that the instability may be a consequence of the misincorporation of bases during this process.

During our previous studies, we observed that the constitutive revertants of gal3 were not normally packaged by )~, and, therefore, permanent transducing lines could not be established (Ahmed and Johansen, 1975). We felt that removal of the chlD-pgl region, which is located between gal3 and the att site for

1 Abbreviation used: kb, kilobase

0026-8925/78/0163/0189/$01.60

190 A. Ahmed and D. Scraba: IS2-Specific Deletions in E. coli

K • E IS2 ch/D pg/ 7t

I~l I I ~ I II II

Deletion L- . . . . . . . . . .~

K T E p 4----- ~ ?~ (hi I I ~ II It

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Fig. l a -d . Scheme proposed to explain the exclusive appearance of constitutive revertants from gal3 strains carrying chlD-pgl deletions. The deletions have a fixed end-point at the left (i.e. at the right terminus of IS2) but variable end-points at the right (a and b). Thus, the deletions retain the IS2 element which is shown to carry a promoter and a rho site. Excision of the IS2 (c) would yield constitutive revertants by fusing the gal structural genes to other promoters at the right. Elimination or alteration of the rho site by imprecise excision, partial deletion, or mutation (d) would allow gal expression from the IS2 promoter. Therefore, both processes would yield constitutive revertants only. Wavy lines with arrow- heads indicate the initiation, direction, and termination of transcription

prophage 2 might facilitate packaging. Unexpectedly, we found that gaI + revertants arising from gal3(2) strains harboring chlD-pgl deletions were nearly always constitutive. Inducible revertants were extre- mely rare. This was surprising because, ordinarily, the gal3(2) strain reverts to produce both inducible and constitutive revertants. This peculiar property of chlD-pgl deletions was particularly conspicuous when the deletions were isolated in the presence of prophage 2. In order to explain the exclusive appearance of constitutive revertants from gal3 AchlD-pgl(2) strains, we proposed that these deletions terminate preferen- tially at (or near) the right extremity of the gal3 insertion, as shown in Figure 1 (Ahmed and Johansen, 1975). During this process, a portion of galOP is removed. It can be seen that (i) excision of IS2 from such deletions would fuse the gal structural genes to other promoters, and (ii) elimination of the rho site by mutations would allow transcription from an IS2 promoter (Rak, 1976; Ahmed and Johansen, 1978). Therefore, all revertants would be constitutive. An expectation of this model is that the chlD-pgl deletions would have a fixed end-point at their left (i.e. at the right end of IS2), while their other end- points would be variable extending into various genes on the right. Reif and Saedler (1975) have also reported deletion specificity for I S / b u t it is not clear from their data whether the deletions extend up to, or remove, the insertion element.

In this communication, we provide direct physical evidence to show that, in a constitutive revertant (gale331) of the gal3 insertion, chlD-pgl deletions ter-

minate specifically at the right terminus of IS2. We also show that the frequency of these deletions is generally increased in constitutive revertants of gal3.

Materials and Methods

General Procedures

The growth media (L-broth, minimal-galactose, tetrazolium-galactose, EMB-galactose, and chlorate plates) were prepared according to the methods described by Miller (1972). Other procedures employed in this work have been described earlier (Ahmed, 1975; Ahmed and Johansen, 1975)

Construction of Strains

All strains were derived from E. coli K12. The chlD-pgl deletions were isolated from strain 331, whose genotype is F galC331 recA1 sUpOm thi strA (2ci857). This strain carries an unstable constitutive reversion (gale331) of the gal3 mutation, which retains the complete IS2 element inserted in orientation I in the galOP region (Ahmed and Johansen, 1978).

Chlorate-resistant mutants, selected by the method of Adhya et al. (1968), were tested for pgl (Kupor and Fraenkel, 1969). The chID-pgl mutants, most of which were deletions, were checked for the retention of 2 (failure to grow at 42 ° and lysis on heat induction) and galC331 (pink colonies producing deep red gal- segregants on tetrazolium plates). In this manner, several gaF331 AchlD-pgl (2) strains were isolated.

Heat-induced 2 lysates from each deletion were used to transduce a gal3 recipient on EMB-galactose. The 2gall2 ratios in these LFT lysates varied from 0.3 x 10 - 7 to 1.9 x 10- 7. The gal + papillae arising by gale331 transduction were tested for the production of HFT lysates for three successive cycles. In this manner, several permanent HFT lines producing 2gal~331 A(chlD-pgl) particles were isolated. The presence of the original galC331 reversion was verified by its segregation pattern and kinase assays. The 2gall2 ratio in these lysates was approximately 6.0 x 10 -3.

Construction of strains producing 2gal + 46.1 and 2gal3 phages, used as the sources of reference DN, has been described (Ahmed and Scraba, 1975). 2galC200 A31(chlD-pgl) carries a stable constitutive reversion (gaF200) of gal3 and a chlD-pgl deletion (A31). Construction of this phage has been described previously (Ahmed and Johansen, 1975).

Phage Preparations

Cells were grown and induced for lysis as described earlier (Ahmed and Scraba, 1975). After lysis, the Mg +÷ concentration was raised to 10 2 M. The phage was sedimented at 18,000 rpm for 31/4 hrs in a Spinco No 21 rotor. The phage pellet was suspended overnight in 0.01 vol. of TMG buffer (0.01 M Tris, 0.01 M MgSO,, and 0.01% gelatin at pH 7.4) at 4 °. The suspension was cleared by low-speed centrifugation, and centrifuged to equilibrium in CsC1 (Jordan etal., 1968). The upper band, which correspondend to 2gal phage in each case, was collected and used for the preparation of heteroduplexes.

Electron Microscopy

DNA heteroduplexes were prepared and mounted for electron microscopy by the formamide technique (Davis et al., 1971). The

A. Ahmed and D. Scraba: IS2-Specific Deletions in E. coli 191

concentration of formamide was 40% in the hyperphase and 10% in the hypophase. Following uranyl acetate staining, the D N A - containing grids were rotary shadowed at 8 ° with 20 A of Pt/C. Micrographs were taken with a Philips EM300 electron microscope operated at 60 kV. Under these conditions, the mean length of 2/2 homoduplexes was 15.39_+0.18 ~tm (=46.5 kb). The lengths of the reference Agal + and 2gal3 D N A molecules are 40.1 kb and 41.2 kb, respectively. Estimates of length are based on measurements of 6 or more heteroduplexes.

Deletion Frequencies

The frequencies of chlD-pgl deletions were determined on the basic gal3 strain (F thr leu lac gal3 thi SUps+m) and its derivatives (Morse, 1967). An inducible revertant ofgal3 was used as the gal ÷ control. The strains gale31, gale33, and gale200 are stable constitutive revertants of gal3. The unstable constitutive revertant gal~331, and the gal operator-constitutive mutat ion 0c81~ (Buttin, 1963) were transferred into the above genetic background by Pl transduction of A303 2, a gal-pgl deletion derived from gal3.

Deletion frequencies were also determined on gal3 and gal~31 strains lysogenized with 2ci857 or its xisl susJ6, int6, and red1 derivatives. 2b2cI was used as helper for the construction of Aint lysogens.

One thousand chlorate-resistant mutants were randomly picked from ten independent overnight cultures of each strain, and tested for the pgl characteristic. Deletion frequencies are expressed as A(chlD-pg[)/ viable cell, and represent typical results obtained from three independent determinations.

Results

Isolation of Deletions

gale331 is an unstable constitutive revertant of gal3 which retains the complete IS2 element inserted in the original polar orientation in the galOP region (Ahmed and Johansen, 1978). Like gal3, it produces chlD-pgl deletions at a relatively high frequency. In addition, it has the advantage that 2gal transducing lines bearing such deletions can be readily isolated. From this strain 1,000 small chlorate-resistant colonies were picked and tested for pgl-. Out of 137 mutants identified as chlD- pgl-, 33 were found to have retained the galC331 reversion and prophage A. Heat-induced lysates from these galC331 chID pgI- (2) strains were used to transduce a gal recipient. Gal + transductants which produced H F T lysates were isolated from 5 of these strains (viz., chID-pgl8, 70, 74, 97 and 124). The transducing particles present in these lysates are referred to as AgalC331 (chlD-pgl). As shown later, all but one of these strains carry extended deletions.

Electron Microscopy of Deletions

The end-points of deletions were determined by electron microscopy of DNA heteroduplexes of 2gale331

A(chlD-pgl) with 2gal + or Agal3. Some representative electron micrographs are presented in Figure 2, and the heteroduplexes are schematically represented in Figure 3. Each heteroduplex shows two loops located towards its left end. The loop closer to the left end is caused by different points of junction of phage and bacterial DNA, and the right loop is caused by the chlD-pgl deletion. The different segments of the heteroduplex were identified from the physical maps of the reference D N A molecules (Ahmed and Johan- sen, 1975). Two points of special interest on the maps of Agal3 and 2gal + DNA (Fig. 3 a and b) are (i) the phage-bacterial D N A junction at co-ordinate 1.9, and (ii) the point of integration of IS2 (1.10+_0.14 kb) at co-ordinate 5.7. The distance from the phage-bacterial junction to the left end of IS2 is 3.8_+0.2 kb, and 4.9+_0.2 kb to its right end. This region includes the gal operon.

First, we consider the heteroduplexes of the deletions with 2gal3. For comparison, these are aligned in Figure 3 (c, d, e, and f) below the 2gal3 map. The left loop, which is normally expected to be a substitution loop, appears as an insertion loop in c, e, and f. The reason is that, due to the presence of chlD-pgl deletions, these Agal genomes have picked up more of each of the phage and bacterial sequences than 2gal3. The position of this loop on the map serves as an accurate marker for the identification of the phage-bacterial D N A junction on the 2gal3 strand. The right loop, in each case, is a true deletion loop caused by the presence of a chlD-pgl deletion on the 2gale331 strand. The lengths of the deletion loops, based on single-strand measurements, are: A97, 5.0_+0.3kb; A70, 10.9+-0.4kb; A74, 10.8_+0.6kb; and A8, 7.2+_0.6kb. Although care was taken to avoid picking the same deletion twice, we cannot rule out the possibility that A70 and A74 represent the same deletion event. Comparison of the location of each of these loops with the Agal3 map indicates that these four deletions were formed very close to the right terminus of IS2. Although the map co-ordinates show some variation, the crucial distance between the two loops (i.e. from the phage-bacterial junction to the right end of IS2) remains constant within experimental error. We take it as evidence to indicate that the chlD-pgl deletions have a fixed end-point at their left, and that this point coincides with the right terminus of IS2. On the other hand, the right end-points of these deletions are variable because three out of the four deletions examined had different lengths.

Further evidence for the conclusion drawn above is provided by heteroduplexes of the deletions with Agal +. If the left end-point of the deletions is indeed fixed at the right end of IS2, then these heteroduplexes are expected to show substitution loops (rather than


Fig. 2a-L DNA heteroduplexes of 2gale331 bearing IS2-specific deletions with 2gal3 and 2gal +. Arrows point to chlD-pgl deletions located at the right end of IS2. a 2ga13/2gal"331 A 74, b 2gal+/2gai~331 A 74, c 2ga13/2galC331 A97, d 2gal+/2galC331 A97, e )~gal+/)~galC331 A 70, and f 2ga13/2galC331 A8

deletion loops) because 2gal + does not ha rbor IS2. One a rm of these loops would cor respond to IS2 of length 1.10 + 0.14 kb, whereas the length o f the other a rm would vary depending upon the size of the deletion. These different heteroduplexes are alig-

ned in Figure 3 (g, h, and i) under the 2gal + map. It is evident that the deletions do fo rm substitution loops, and the left extremity of each loop coincides closely with the point o f integration o f IS2 in 2gal +. In each case, the shorter a rm can be identified as


(a)

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Fig. 3 a-k. Schematic representation of heteroduplexes. All distances and map co-ordinates are expressed in kilobases. The D N A sequences shown are: (---) phage sequence; ( 7 IS2; and ( ~ , ~ ) bacterial sequence. The physical maps of a Agal3 and b 2gal + reference D N A are shown at the top. The location of some genetic loci is indicated below the maps. Heteroduplexes of Agal3 with 2gale331 bearing c A97, d A70, e A74, a n d f A8 are aligned, for comparison, below the Zgal3 map on the left. In each case, the upper line represents 2gal3 strand and the lower line represents 2gale331 A(chlD-pgl) strand. Note that the size of the deletion loop (indicated by slanted arrows) varies but its location remains fixed. Heteroduplexes of 2gal + with AgalC331 bearing g A97, h A70, and i A74 are shown on the right below the Agal + map. In each heteroduplex, the upper line represent 2gal + strand, and the lower line represents ZgaF331 A(chlD-pgl) strand. Only the left portion of each heteroduplex is shown. The exact points of junct ion of phage and bacterial D N A in the left loops are not known for any of these deletions, except A 70. j A 2gal3/2galC200 A31(chID-pgl) heteroduplex showing the location of two independent deletion events (gale200 and A31) near left and right ends of IS2. The substitution loop is believed to be formed by fusion of the two loops, as shown in k on an enlarged scale

IS2 from its size and location. The measurements for the shorter arm are: A97, 1.09_+0.15 kb; A70, 1.00_+0.13 kb; and A74, 1.26 _+0.14 kb. Therefore, no part of the IS2 element is apparently deleted. The length of the other arm, which corresponds to the chlD-pgl deletion, varies and is generally in agreement with the lengths estimated from 2gal3 heteroduplexes. These measurements are: A97, 4.9_+0.3kb; A70, 10.3_+0.5kb; andA74, 10.9+_0.8kb. In each case, the distance between the two loops (i.e. from the phage-bacterial D N A junction to the left end of IS2) remains constant within experimental error. There- fore, these deletions seem to conform, in every respect, to the kind postulated in Figure 1.

Location of the Rho-Sensitive Site on IS2

We have shown previously that a stable constitutive revertant (gaF200) of gal3 arose by a partial deletion

(approx. 4/s ) of the IS2 element, presumably by removal of the site of action of the rho factor (Ahmed and Johansen, 1975). Heteroduplexes of 2gaF200 (which also carried a chlD-pgl deletion 31) with Agal + exhibited a substitution loop in which the short arm (0.23 +0.03 kb) represented the remaining fragment of IS2, and the long arm (6.3_+0.4 kb) was due to the chlD-pgl deletion 2. We have now studied heteroduplexes of AgaF200 A31(chlD-pgl) with 2ga13 and found a similar substitution loop, as shown in Figure 3j. The fact that a substitution loop, rather than a deletion loop, is formed indicates that the 0.9 _+ 0.1 kb

2 In a previous paper (Ahmed and Johansen, 1975), we had considered an alternative interpretation for the configuration observed in Zgal+/2gal"200 A31(chID-pg 0 heteroduplez}es. Accord- ing to that scheme, the ga#200 reversion arose by the inversion of IS2 (Saedler et al., i974) followed by the chID-pgl deletion which extended into, and removed, approximately 4/5 of the IS2. In view of the deletion specificity demonstrated in the previous section, this interpretation can be ruled out


segment deleted in galC200 was derived f rom the left part of the IS2. If the reversion had removed a segment f rom its right, a deletion loop would have been formed. Therefore, the 7 .5+0.5 kb arm of the loop was apparently formed by the coalescence of two separate loops, viz., a deletion loop caused by gal~200 located near the left end of IS2, and a larger deletion loop caused by A31 located at its right (Fig. 3k). This result implies that the site of rho action is located on the left 4/s port ion of the IS2, and the right 1/5 fragment remaining in gaF200 is sufficient to confer end-point specificity on chlD-pgl deletions.

A chlD-pgl Substitution

During this study an exceptional mutant 124 was found which had originated by the substitution of a D N A segment from the chlD-pgl region by a short piece of foreign DNA. The 2gal+/2galC331 subs. 124(chlD-pgI) heteroduplexes show three loops. F rom the left end, the first loop was caused by different junctions of phage and bacterial DNA, the second loop was IS2, and the third loop was caused by the 124 substitution. A 5.9_+0.4 kb segment f rom the chlD-pgl region was removed and substituted by a 0.5 + 0.1 kb segment of unrelated DNA. This genetic exchange, which is probably a reciprocal transloca- tion, occurred at a distance of 3.3 kb f rom the IS2 and bears no resemblance (except for the phenotype) to the deletions described above.

Table 1. Deletion frequencies of constitutive revertants of gal3 and the influence of prophage 2

Genotype Chlorate-resistant A (chlD-pgl) A (chlD-pgl) mutants per among chlorate- per viable cell resistant mutants viable cell ( × lo 6) (%) ( x lO ~)

gal + 2.1 0.05 0.1 g al÷ ()0 4.1 0.1 0.4 gal3 15.9 0.6 9.5 gal3() 0 5.4 1.4 7.6 gale200 4. i 1.2 4.9 gale200(2) 3.9 5.3 20.7 galC31 4.4 3.4 15.0 galC31(2) 3.9 6.8 26.5 galC33 9.7 3.0 29.1 gal~33(2) 6.0 4.8 28.8 gal~331 3.3 3.3 10.9 galC331()O 3.0 2.4 7.2 galO~81-2 2.4 1.1 2.6 galO~81-2()O 3.1 0.2 0.6

AI1 strains are isogenic derivatives of the basic gal3 strain. Their origin or construction is described under Materials and Methods. The designations gal ÷ and galc refer, respectively, to inducible and constitutive revertants of gal3

constitutive revertants of gal3, and might be related to the occurrence of transcription along IS2.

The recA mutation had no effect on the frequency or specificity of the IS2-specific deletions. The int, xis, and red mutations of the prophage had no appreciable effect either (data not presented).

Influence of Constitutive Reversions on Deletion Frequency

Discussion

Characteristics of Deletions

We reported previously that the frequency of spontaneous chlD-pgl deletions is increased in the presence of the gal3 insertion, and that the end-point specificity is influenced by prophage 2 (Ahmed and Johansen, 1975). During the present study, it appeared that these frequencies were even higher in constitutive revertants ofgal3. Hence, we undertook an examination of deletion frequencies in different revertants, both in the presence as well as the absence of 2. The results (Ta- ble 1) show a general increase in the frequencies of deletions in stable constitutive revertants of gal3 (viz., gale31, galC33, and galC200), particularly in the presence of prophage 2. The increased frequencies are not caused simply by the occurrence of transrip- tion, because an operator-constitutive mutat ion (gal0~81-2) does not show such an increase. Similarly, the deletion frequency of a gal ÷ strain was not influenced by growth in the presence of the inducer D-fucose. Hence, this phenomenon seems unique to

We have studied the properties of deletions formed in response to IS2. Although a constitutive revertant (gale331) of the gal3 mutat ion was used in the present study, we believe our conclusions are equally applica- ble to the original IS2 element with respect to deletion formation. The main conclusions can be summarized as follows :

(i) The presence of IS2 in the galOP region causes an increase in the frequency of chlD-pgl deletions (Table 1).

(ii) The left end-point of these deletions is fixed, being located at (or near) the right terminus of IS2 (Figs. 2 and 3). The complete insertion element is apparently retained.

(iii) The right end-points of the deletions are variable and exhibit no apparent specificity. The sizes of the five deletions studied were: A97, 5.0 kb; A70, 10.9 kb; A74, 10.8 kb; AS, 7.2 kb; and A31, 6.3 kb. This conclusion is supported by genetic tests reported


earlier (Ahmed and Johansen, 1975) which show that the IS2-specific deletions extend randomly into various genes at the right.

(iv) End-point specificity is a direct consequence of the presence of IS2, because no such specificity is observed in the absence of this element (Ahmed and Johansen, 1975).

(v) This specificity is enhanced by the presence of prophage )~, but int, xis, and red functions are apparently not involved.

(vi) The complete IS2 element is not needed for the expression of end-point specificity. A constitutive revertant (galC200), which retains only 230 base pairs derived from the right end of IS2, can produce a similar deletion (Fig. 3j and k). Therefore, the basis of specificity appears to reside in a base sequence located near the right end of IS2.

(vii) Deletion frequency is increased in constitutive revertants derived from IS2. This effect is highly specific because it is observed in constitutive revertants of gal3 but not in an O ~ mutation (Table 1).

(viii) A further increase in deletion frequencies of some constitutive revertants (e.g. gal~200) is observed in the presence of prophage 2. However, this effect is not general.

Mechanism of Deletion Formation

The mechanism envisioned for the formation of spontaneous deletions in response to IS2 (Ahmed, 1977) is as follows. A specific base sequence located at, or near, the ends of IS2 is recognized and acted upon by a restriction-kind of endonuclease. The initial nick is enlarged by unidirectional, exonucleolytic degradation to various extents. Such a process would lead to the formation of deletions of varying lengths extending outwards from one end-point fixed at IS2. Davis and Parkinson (1971) have proposed a similar explanation for the int-promoted deletions of 2. In- volvement of nicks in deletion formation is suggested by experiments using a temperature-sensitive D N A ligase mutation (A. Ahmed, manuscript in preparation). The frequency of chlD-pgl deletions in a gale331 lig ts7 strain is increased 25-fold at a moderately re- strictive temperature, indicating that nick accumula- tion may provoke deletion formation. The cellular and prophage functions involved in the formation of IS2 deletions have not been identified. Since these deletions can be formed in the absence of ), it appears that the sequence-specific endonuclease is coded by the host cell or IS2. The role of prophage 2 may be additive, i.e. through a function (or process) which facilitates deletion formation.

The data in Table 1 show that the frequency of IS2-specific deletions is generally increased in stable

constitutive revertants of gal3. These revertants arise by partial deletions of IS2, and characteristically retain a part of it (Ahmed and Johansen, 1975). It seems that only the frequency, but not the specificity, of deletions is affected. We suggest that this increase in deletion frequency is caused by the un- masking of the nicking site on IS2 during transcription. IS2 carries a strong promoter acting in orientation II (Saedler et al., 1974), which corresponds to right- ward transcription in Figure 1. Perhaps the activity of this promoter is enhanced in constitutive revertants. Elsewhere, we have proposed that two other phenomena associated with IS2, viz., the instability of constitutive revertants and the failure to form 2gal particles bearing constitutive reversions, may also be caused by frequent nicking of IS2 ends during transcription (Ahmed, 1977; Ahmed and Johansen, 1978).

Electron microscopy does not enable us to state precisely whether the deletions extend up to, or pen- etrate, IS2. Removal of less than 50 base pairs would ordinarily go undetected by this technique. The length measurements suggest that the IS2 remains intact in at least A97, A 70, and A 74. However, it may be impor- tant to point out that gal3(2) strains bearing IS2- specific chlD-pgl deletions always show a significant reduction in their reversion frequencies (A. Ahmed, unpublished results). This reduction suggests that the nicking site, where the process of deletion formation is apparently initiated, is located inside IS2 so that the deletions actually remove a few bases from this element. As a result, IS2 is no longer excised effi- ciently. Alternatively, the nicking site might be located precisely at the end of IS2 but the adjacent chromosomal sequence may play in role in the excision process. Hence, removal Of this sequence by deletion may influence excision efficiency. We favor the second possibility because it assigns the specificity of nicking, required for deletion formation, to the same enzyme which carries out the highly accurate processes of IS2 excision and integration. This also implies that integration of IS2 elements into the chro- mosome may not be a totally random process, and that chromosomal sequences may also be recognized.

Acknowledgements. We thank Danielle Macbeth and Roger Bradley for excellent technical assistance. We also thank Dr. S. Adhya for supplying many phage stocks. This work was supported by grants from the National Research Council and the Medical Re- search Council of Canada.

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Ahmed, A. : Mechanism of reversion of the gal3 mutation of Es- cherichia coli. MoIec. gen. Genet. 136, 243-253 (1975)

Ahmed, A.: The gal3 mutation of E. coll. In: DNA insertion elements, plasmids and episomes (Bukhari, A., Shapiro, J., Ad- hya, S., eds.), pp. 37-48. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory 1977

Ahmed, A., Johansen, E.: Reversion of the gal3 mutation of Es- cherichia coli: Partial deletion of the insertion sequence. Molec. gen. Genet. 142, 263-275 (1975)

Ahmed, A., Johansen, E.: The basis of instability of a revertant of the gal3 insertion of E. coli. J. molec. Biol. (in press)

Ahmed, A., Scraba, D. : The nature of the gal3 mutation of Escheri- chia coli. Molec. gen. Genet. 136, 233-242 (1975)

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C o m m u n i c a t e d by O. S idd iq i

Received January 15, 1978

nature of deletions formed in response to is2 in a revertant of the gal3 insertion of e. coli

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