Identification of an Rts1 DNA fragment conferring temperature-dependent instability to vector plasmids

PLASMID 13, 88-98 (1985)

Identification of an Rtsl DNA Fragment Conferring Temperature- Dependent Instability to Vector Plasmids

NORIYUIU OKAWA, HISASHI YOSHIMOTO, AND AKIRA KAJI

Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received May 14, 1984; revised August 10, 1984

The multiphenotypic drug resistance factor Rts 1 expresses a temperature-dependent instability characteristic. This plasmid was digested with the restriction enzyme BarnHI. A DNA fragment with a molecular weight of 5.6 MDa (the H fragment) was inserted into plasmid pBR322 (pFK896) or into pSClO5 (pYH 156) at the BarnHI site. These plasmids were unstable at 42°C but stable at 32’C. A restriction-enzyme map of the H fragment was constructed and the instability phenotype (Tdi) was localized to a DNA fragment with 0.5 MDa molecular weight. The temperature-dependent loss of the unstable plasmid pFK896 is abrupt and no gradual plasmid loss of this multicopy recombinant plasmid is observed. The possibility that the Tdi phenotype is due to overgrowth of R- cells was eliminated. 0 1985 Academic press, I~C.

Rtsl is a multiphenotypic drug resistance factor which confers kanamycin resistance (Km’) (36,15) to bacteria. This plasmid is peculiar in that it confers temperature-sensitive growth to bacteria (Tsg) (10,18). In addition, the Rtsl containing bacteria restrict T4 phage growth at 32°C but not at 42°C (I 7,18). The replication of this plasmid at 42°C apparently involves unique noncova- lently closed circular (CCC) intermediates which can be converted to the CCC form upon temperature shift to 32°C (Tsc) (40). Furthermore, this plasmid exhibits temperature-dependent instability (Tdi), this phenotype was called Insts in our past publication (18). In other words, the plasmid was eliminated when dilute cultures of bacteria containing Rtsl are incubated at 42°C.

the vector pBR322 (pFK896) or pSClO5 (pYH156) and was shown to exhibit the typical temperature-dependent instability. The fragment responsible for this phenotype has been located to a region with a molecular weight of 0.5 MDa.

MATERIALS AND METHODS

Bacterial strain and plasmids. The Esche- richia coli strain 20S0 (6,ll) was used in our previous communication (8). The plasmids are listed in Table 1.

Media. TSB (Trypticase Soy Broth) (BBL) and L-broth (LB broth base) (GIBCO for transformation experiment) were employed as liquid media. MacConkey agar base (DIFCO, without lactose) containing 1% glucose was used as plating medium.

Earlier, we had digested Rtsl with restric- Biochemicafs. Ampicillin sodium salt (Ap, tion enzymes and created a mini-Rtsl in an 100 fig/ml), chloramphenicol (Cm, 50 pg/ attempt to localize the portions of DNA ml), kanamycin sulfate (Km, 50 &ml), and which are responsible for these phenotypes tetracycline hydrochloride (Tc, 25 &ml) (39). Miniplasmids which still confer Tsg and were from Sigma. Endonucleases AvaI, Tsc have been obtained (S. Finver et al., in BamHI, EcoRI, and Hind111 were from BRL; preparation). In this communication, we have NruI and T4 DNA ligase were from New focused our attention on the temperature- England Biolabs; PvuI and PvuII were from dependent elimination of Rtsl plasmid. A Boehringer Mannheim. DNA molecular 5.6MDa BamHI fragment of Rtsl designated markers, X HindIII (7,30) and $X 174 Hue111 as the H fragment was cloned into (12), were from New England Biolabs.

0147-619X/85 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reprcducl~on in any form resewed.

88

TRANSFER OF Rtsl EXPRESSION TO PLASMIDS 89

TABLE 1

PLASMIDS

Plasmids

Rtsl

pBR322

pBR328

pSClO5

pFK896 pYH156

pYH65

pYH74”

pYH81 o

pYH98“

pYH157”

pYH212”

pYH217”

pNK2

pNK7’

pNK28’

pNK33 (143)’

pNK98”

pNK100”

pNK108”

pNKll1”

pNKll6”

pNK124’

pNK137”

Character or fragments

Naturally occurring plasmid carrying Km’

E. coli plasmid carrying Ap’, Tc’

E. coli plasmid carrying Ap’, Tc’ and Cm’

E. coli plasmid carrying Km’, Tc’

The H fragment of Rtsl and pBR322 (BarnHI site) The H fragment of Rtsl and pSClO5 (BumHI site)

a, b, d, e, f, g, h

g> h

a, b, c, d X 2, e X 2, f X 2, g X 2

a, b, d, e, g, h

a, b

a, b, g X 2, h X 2

a X 2, b X 2, g, h

The H fragment of Rtsl and pBR328 (BarnHI site)

a, b, g

is

a, g

a’

a

b

b’

b, g

b’, g

a’, g

Reference or source

(36, 38)

(4, 34, 38)

E. Lcderberg, (32)

E. Lederberg, (20)

This paper, Figs. 1, 3 This paper, Fig. 1

This paper, Fig. 4

This paper, Fig. 4

This paper, Fig. 4

This paper, Fig. 4

This paper, Fig. 4

This paper, Fig. 4

This paper, Fig. 4

This paper, Table 2

This paper, Fig. 5

This paper, Fig. 5

This paper, Fig. 5

This paper, Fig. 5

This paper, Fig. 5

This paper, Fig. 5

This paper, Fig. 5

This paper, Fig. 5

This paper, Fig. 5

This paper, Fig. 5

0 The plasmid contains fragment(s) from the H fragment and pBR322 (Ap’) with deletions.

Isolation of plasmid DNA, restriction-enzyme digestion, and pur$cation of DNA fragments. All plasmids were isolated from overnight cultures by the cleared lysate method and ethidium bromide-cesium chloride cen- trifugation as previously described (38). Con- ditions for restriction enzyme reactions were as specified by the suppliers. The digests were separated by agarose gel electrophoresis, and the fragments were electroeluted (22). They

were then concentrated and purified by Elu- tip-d (Schleicher & Schuell) and ethanol pre- cipitation.

Ligation and bacterial transformation. DNA fragments obtained by restriction en- donuclease digestion were mixed with a salt mixture to obtain a final concentration of 10 mM MgC12, 25 mM NaCl, 1 mM ATP, 10 mM dithiothreitol, and 50 mM Tris-HCl (pH 7.5). T4 DNA ligase (2 units) was added and

90 OKAWA, YOSHIMOTO, AND KAJI

samples were incubated at 15°C for 16 h. Competent cell cultures were prepared and transformed by the calcium chloride procedure as described (21). Transformation mix- tures were diluted with L-broth and incubated at 32°C for 1 to 1.5 h. Transformants were selected on MacConkey agar plates containing 1% glucose and antibiotics.

Agarose gel electrophoresis. DNA samples were subjected to electrophoresis through OS- 1.5% agarose in 89 mM Tris-HCl (pH 8.3), 2.5 mM EDTA, 89 mrvt boric acid, and ethidium bromide (0.5 gg/ml), at a constant voltage (40 V) for 15 h.

Tdi activity test. Bacteria were first plated on MacConkey agar base containing 1% glucose (supplemented with 100 pg/ml ampicillin) and an isolated colony was used. In some cases frozen cultures (overnight growth with Trypticase Soy Broth (TSB) with ampicillin which had been stored at -70°C) were inoculated into 10 ml of TSB. Cultures were divided into two equal parts and they were separately incubated at 32 and 42°C respectively, for 20 h with shaking. They were then inoculated into a fresh 5 ml of TSB and further incubated for 16 h. Samples were then diluted and spread onto MacConkey agar 1% glucose and grown overnight at 32°C. Each plate was replica plated onto MacConkey agar 1% glucose, with and without ampicillin, and grown overnight at 32°C. Tdi activity was expressed as the percentage of ApS colonies; all values represent an average of five or more experiments.

RESULTS

The H Fragment of Rtsl Confers Temperature-Dependent Instability (Tdi) to Vector Plasmids

Sixteen DNA fragments (A to P) were obtained by the digestion of Rtsl DNA with the restriction enzyme BarnHI. The sum of the molecular masses of these fragments is approximately 127.5 MDa. Since the molecular mass of Rtsl is 126 MDa (9), these fragments account for most, if not all, of the

of these fragments (except for the G fragment) into the vector plasmid pBR322 (18). Of these plasmids, pBR322 containing the H fragment (5.6 MDa) was designated as pFK896 which was isolated in our laboratory by S. Finver and T. Yamamoto through the method described in a previous paper (39). In a similar manner, this fragment was cloned into pSClO5 at the BamHI site (pYH156).

Recombinant plasmids composed of vec- tors and the H fragment are unstable at 42°C but are less so at 32°C (Fig. 1). For example, only 20% of the bacterial population lost pFK896 at 32°C within 40 generations; whereas, the same plasmid was eliminated completely at 42°C in less than 40 generations. Similarly, pYH 156 is mostly lost within 40 generations at 42°C; however, this plasmid is relatively stable at 32°C (approximate 30% loss). Under identical conditions, the control plasmids, pBR322 and pSC105 showed complete stability at both temperatures.

The Loss of Plasmid Due to the Presence of the H Fragment Is Not Caused by Overgrowth of R- Cells

It appeared possible that, if plasmids with the H fragment retard the growth of bacteria,

J 42%

20 40

Generations

FIG. 1. Time course of loss of the Tdi plasmids. Escherichia coli 20S0 harboring the plasmids were inoculated at a density of 2 X 103/ml in Trypticase Soy Broth. Samples were taken at various times, and antibiotic (ampicillin or kanamycin)-resistant colonies and total number of colonies were counted. pFK896 (Tdi-pBR322, A); pYH156 (Tdi-pSClO5, A); pBR322 (0); pSClO5 (0). BumHI digestion of pFK896 and pYHl56 resulted in the H fragment (5.6 MDa) and its vector DNA (2.7,

Rtsl DNA. We have been able to clone most 10.5 MDa, respectively) by agarose gel electrophoresis.


R- cells which were formed by loss of the plasmid might overgrow the entire population and give the appearance that the plasmid itself is unstable. To examine this possibility, we used pBR328 to construct a new plasmid derivative. This vector has a Cm’ gene and the BarnHI site which can be used for inser- tion of the H fragment. The resulting plasmid, designated pNK2, has Ap’, Cm’, and Tc”. If bacteria are incubated in the presence of Cm, those cells which lost the plasmid would not be able to grow because of the presence of Cm. On the other hand, due to the bacteriostatic nature of Cm action, these cells remain viable in the culture. Thus, after incubation of cells containing pNK2, the presence of R- cells would indicate that plasmid was indeed lost from them and they are not the result of rapid growth of R- cells.

As shown in Table 2, approximately 22% of the entire population loses pNK2 per one division time at 42°C. We, therefore, concluded that the appearance of R- cells is due to loss of plasmid and not due to overgrowth of R- cells.

LOSS of the Tdi Plasmid Is Not Gradual

Since pBR322 is a multicopy plasmid (5), one expects that its derivative pFK896 is also

a multicopy plasmid. It is, therefore, conceiv- able that the loss of this plasmid may be gradual. In other words, one may be able to observe cells harboring some plasmids but not as many as usual [24 copies per cell (5)]. It has been shown that the level of Ap’ is proportional to the number of Ap’ plasmids contained within a cell (37). Therefore, it is possible to examine whether or not the loss of plasmid is gradual, by measuring the level of ampicillin resistance during growth of the cells at 42°C. In the experiment indicated in Fig. 2, cells harboring pFK896 were grown at 42°C for various periods (0, 5, 20, 32, and 40 generations). At these intervals, cells were plated to medium containing various amounts of ampicillin. If cells containing a reduced number of these plasmids are pro- duced, one expects that they would exhibit an intermediate level of resistance. This would produce a “slope” rather than a “step” in the figure which plots percentage of viable count against concentration of ampicillin in the testing plates. If the loss is gradual, but occurs synchronously rather than randomly, one expects a series of steps with drops at smaller concentrations. It can be seen from Fig. 2 that at each interval, the level of Ap’ is always the same among the various popu-

TABLE 2

THEL~SS OF Tdi RECOMBINANTPLASMIDISNOTDUETOTHEOVERGROWTHOFR-CELLS~

Time (hd Bacterial/ml

32°C 42°C

pBR328 PNK~~ pBR328 PNIQ

A0 total B 17 total co AP’ D 17 AP’

EC probability (W) of plasmid loss

2.3 x 10’ 1.2 x lo4 2.3 X lo3 1.2 x lo4 4.5 x 109 1.8 x lo9 4.1 x 10s 9.1 x lo8

0 0 0 0 0 5.5 x lo6 0 2.0 x lo*

0.0 0.3 0.0 22.0

’ E. coli 20S0 harboring the plasmids were inoculated into Ttypticase Soy Broth containing Cm (IO&ml), and incubated at 32 or 42°C. Samples were taken at 0 time and after 17 hr growth, and Ap’ and total numbers of colonies were counted.

’ The orientation of the H fragment in pNK2 is the same as in pFK896: Plasmid DNA was digested with EcoRI, and the 5.62- and 3. I-MDa bands were obtained.

‘E Values were probability (W) of plasmid loss of Apr cell/each cell division. E values were calculated from the formula: E = (D - C)/(B - A)X 100 [B%- A;(D - C)/(B)X 1001.


loo loo Gwwrlions Gwwrlions

pBR322

. ' W25 a05 Cl2 1.0 IS.0 6.0 0 0.025 0.1 0.5 2.5 IQ0 20.0

Ampicillin mglml

FIG. 2. The level of ampicillin resistance of E. coli with the Tdi-pBR322 (pFK896) during growth in the absence of ampicillin at 42°C. E. coli harboring the plasmids were grown in Trypticase Soy Broth without antibiotics. At various times, samples were transferred to plates containing various amounts of ampicillin as shown in the figure. The colonies grown on ampicillin plates are expressed as relative numbers to those grown on plates without ampicillin.

lations and therefore, the curve is a “step” rather than a “slope. ” “Drop” of the step was at 10 mg of ampicillin per ml regardless of period of incubation. It appears, therefore, that the loss of plasmid is sudden and no gradual decrease of plasmid number takes place. If copy loss occurred randomly during cell division, the probability of simultaneous curing would be approximately 1/224 (5.9 x lo-*) (26).

Restriction Enzyme Map of pFK896

Since the H fragment is relatively large (5.6 MDa), we attempted to narrow down the region of Tdi activity. The first step was to construct a restriction enzyme map of this plasmid (pFK896). We subjected this plasmid DNA to digestion with the restriction enzymes, AvaI, NruI, HindIII, EcoRI, BamHI, PvuI, and PvuII. The results are summarized in Fig. 3. As shown in this figure, the H fragment was cut into small fragments a through h.

Localization of Tdi Characteristic to the b Fragment

In order to assign the Tdi characteristic to a smaller region of the H fragment, we have constructed various derivatives of pBR322 containing portions of the H fragment as shown in Fig. 4. The orientation of each fragment in relation to pBR322 is shown in Fig. 4. The derivative plasmids were tested for the Tdi activity and the results are summarized in Table 3. It is clear from Table 3 that pYH217, 212, 98, 81, and 65 are Tdi positive, while the rest of the derivatives are Tdi negative. By comparing Fig. 4 and Table 3, we made a tentative conclusion that the Tdi character needs the three fragments a, b, and g. In addition, the orientation of these DNA fragments in relation to pBR322 DNA appears to have no effect on the expression of the Tdi characteristic. However, the re- quirement of the fragments a, b, and g was deduced from physiological characterization of derivative plasmids which contain these three fragments in addition to other frag-

Rtsl EXPRESSION TO PLASMIDS 93 TRANSFER OF

Pvu It Bam H’ , I NW I

ECORI Hmd III

am HI

FIG. 3. Restriction map of pFK896. The numbers in parentheses below represent the molecular masses (MDa) of DNA fragments obtained by the restriction enzyme(s) digestion of pFK896 and its derivatives. NruI (3.30,2.70, 2.05), PvuI (8.40), EcoRI (4.80, 3.50), Hind111 (3.65, 3.65, 0.86), AvaI (4.30, 3.05, 0.93). NruI and PvuI (3.25, 2.10, 1.80, l.lO), AvuI and PvuI (4.20, 1.65, 1.50, 0.92), AvuI and Hind111 (1.95, 1.85, 1.60, 1.25, 0.92, 0.86), AvaI and EcoRI (2.60, 1.95, 1.65, 1.25, 0.92), EcoRI and NruI (2.18, 2.15, 2.05, 1.35, 0.68), BumHI and Hind111 (3.50, 2.50, 1.20, 0.87, 0.21) PwII (2.35, 2.10, 1.25, 0.76, 0.60, 0.60, 0.26), PvuII and Hind111 (2.05, 1.50, 1.25, 0.76, 0.72, 0.60, 0.50, 0.36, 0.26); pYH74, BumHI and PvuII (1.65, 1.15, 0.80, 0.24); pYH65, BarnHI and PvuII (2.52, 1.77, 1.19, 0.81, 0.62, 0.53, 0.24); pYH65, PvuII (2.52, 2.30, 1.35, 0.81, 0.64).

ments. This leaves the possibility that fragments other than a, b, and g may influence them in the expression of the Tdi phenotype.

To further elucidate the role of these three fragments, the next series of experiments were performed. As before, various derivatives of pBR322 containing a, b, and g were constructed as shown in Fig. 5. The Tdi activity of these plasmids as are shown in Table 4. As one would expect from the conclusion obtained from Table 3, pNK7, which contains a, b, and g gave a positive Tdi characteristic. Plasmids containing the a or g fragment alone were Tdi negative. Sur- prisingly, however, plasmids with only the b fragment or b’ fragment (pNK108 and pNK Ill, respectively) were remarkably positive for the Tdi characteristic. The b’ frag-

ment, which is smaller than the b fragment, had a strong Tdi characteristic. It is possible that this may have something to do with the presence of an additional pBR322 fragment in this plasmid as indicated in Fig. 5. On the other hand, pYH157 (a and b), pNK116 (b and g), pNK124 (b’ and g), pNK143 (a and g), and pNK137 (a’ (a and part of b) and g) were Tdi negative. We suggest that the a or g fragment may suppress the Tdi activity of the b (or b’) fragment, while the a and g fragments together cannot suppress Tdi activity of the b fragment (pNK7).

DISCUSSION

In an attempt to elucidate the temperature- dependent instability (Tdi) of Rtsl, we have identified a BarnHI fragment of 5.6 MDa (the H fragment) which is responsible for this phenotype. Upon further digestion of this fragment, a smaller fragment (0.5 MDa) was identified as the major fragment causing Tdi. The Tdi phenotype expressed by these fragments is greatly influenced by the presence of other DNA fragments. For example, it is not clear why pYH156 (pSC105 and H) is lost less rapidly than pFK896 (pBR322 and H). In addition, in the preliminary attempt to identify tdi fragments, it appeared as if three fragments a, b, and g were responsible for this phenotype. However, upon further examination (Table 4) it turned out that the a as well as the g fragment inhibit expression of the Tdi phenotype by the b fragment only. These inhibitory effects of a and g apparently are counteracted by each other. In a similar manner, one can explain the phenotype which we identified as Ins expressed by the BumHI fragment of Rtsl with a molecular mass of 1.3 MDa (the L fragment) (18). This fragment by itself appears to confer temperature-inde- pendent instability to a vector plasmid such as pBR322. Since there is no instability observed with Rtsl at 32°C this phenotype of the L fragment is created as an artifact of isolation. Apparently, this effect of ins was nullified by the presence of other DNA fragments in Rtsl.


FIG. 4. Restriction enzyme map of derivatives of pFK896. The arrow shows the orientation of each fragment (a clockwise direction represents the same orientation as in pFK896). Restriction enzyme maps are determined in the same way as described in Fig. 3. E, EcoRI; H, HindIII; B, BumHI; N, NruI; P, PvuII; A, AvaI. Shadowed areas represent pBR322 fragments. pYH65, Aval (4.30, 3.00), BumHI (4.60, 2.60); pYH74, Hind111 (3.65); pYH81, BumHI (9.40), AvaI and EcoRI (2.55, 2.55, 1.75, 1.20, 0.95), EcoRI (5.50, 3.80), AvaI (4.60, 3.80, 0.95), Nrul (5.60, 2.60, 1.25); pYH98, AvaI and Hind111 (1.85, 1.75, 1.58, 1.23), AvuI and BarnHI (3.30, 1.40, 1.00, 0.71), NruI (2.85, 2.10, 1.25); pYH157, AvaI (3.05); pYH212, AvnI (3.70, 3.05), BamHI (3.75, 1.60, 1.32); pYH217, AvaI (3.05, 2.98); BamHI (3.10, 1.60, 1.35).

One can ask the question whether or not all of the Tdi activity of the Rtsl plasmid can be accounted for by the b fragment. The degree of instability of pFK896 (pBR322 containing the H fragment) is greater than that of pNK108 (which contains the b frag-

TABLE 3 LOCALIZATION OF Tdi ACTIVITY’

Loss of plasmids (%)

Plasmids 32°C 42°C DNA fragments

pFK896 pYH65 pYH74 pYH8 1

pYH98 pYH157 pYH212 pYH2 I7

2.6 100.0 20.5 54.5 0.0 7.5

34.0 100.0

19.4 92.5 0.0 0.0 0.0 59.3

39.0 100.0

a, b, c, d, e, f, g, h a, b, d, e, f, g, h g, h a, b, c, d X 2, e X 2,

fx2,gx2 a, b, d, e, g, h a, b a, b, g X 2, h X 2 a X 2, b X 2, g, h

d The procedure for determining instability is described under Materials and Methods.

ment) or pNKll1 (containing the b’ fragment). These results suggest that the Tdi phenotype may be strengthened by the other fragments existing in the H fragment. Indeed, in our preliminary experiments we have oc- casionally observed weak Tdi phenotypes expressed by groups of fragments such as d and e or d, e, and c as well as h of the H fragment (unpublished observation). How- ever, the Tdi b-fragment phenotype is by far the most pronounced among Tdi derivatives of the H fragment.

In a similar fashion, we may ask whether the H fragment is the only fragment which is responsible for the temperature-dependent instability of Rtsl. It has been reported that the replication fragment of Rtsl confers the Tdi phenotype (19). We have cofirmed that observation in our system, where pSClO5 containing the D fragment (BumHI fragment containing the replication origin, 15.9 MDa) was found to be unstable at 42°C. The degree of instability of this plasmid was greater than pFK896 (unpublished data). However, in


FIG. 5. Restriction enzyme map of plasmids containing the a, b, and g fragments. Restriction enzyme maps are determined in the same way as described in Fig. 3. E, EcoRl; B, BarnHI; H, Hindlll; P, Pvull; N, Nrul; A, Avul. Shadowed areas represent pBR322 fragments. pNK7, EcoRI and Hind111 (3.05, 0.22); pNK28, EcoRl and Hind111 (2.70, 0.22); pNK98, BumHI and Pvull (1.66, 0.52); pNK100, BumHI and Nrul (2.33, 0.40); pNK108, Avul and Nrul (2.42, 0.60); pNKll1, Avul and Z-Vu11 (3.10, 0.52); pNKl16, EcoRl and Hind111 (3.00, 0.22); pNK124, EcoRl and Hindlll (3.68, 0.22); pNK137, EcoRl and Hind111 (2.15, 0.22); pNK143 (pNK33), EcoRl and Hindlll (2.71, 0.22). The b’ fragment extends from Pvull site to Avul site of the b fragment. The a’ fragment includes the a fragment plus from Nrul site to Pvull site of the b fragment.

these studies, neither we nor others (19) have eliminated the possibility that the apparent loss of plasmid from the host bacteria may be due to the overgrowth of bacteria which have spontaneously eliminated their plasmids. In fact, such a possibility should be considered seriously because the temperature-sensitive growth phenotype (Tsg) resides in Rtsl . We ruled out, through the use of the bacteriostatic inhibitor chloramphenicol, this possibility of elimination of plasmid due to overgrowth of R- cells (Table 2).

As for the stability of plasmids in bacteria, a number of workers have identified plasmid DNA elements which are responsible for the stable maintenance of plasmids. These frag-

ments of DNA are often called the par (or stb) region of the plasmid (1,13,14,23,24, 27,29,31,35). It should be pointed out that there exists an important difference between the concept of par and ins. The par region is the fragment of DNA which positively contributes toward the stability of plasmids. In contrast, the ins DNA fragment negatively contributes toward the stability of plasmids. Other examples of negative elements for stability have been reported with artificially created mutated fragments of DNA (16). The H fragment is the first example of a naturally occurring DNA which clearly contributes negatively toward the maintenance of other vector plasmids at 42°C.

TABLE 4 may possibly act in cis as we proposed for INTERACIXON OF a, b, AND g FOR Tdi ACT~VITY~ Ins (18). Further studies on the mechanism

of instability of the b fragment are in progress Loss of plasmids with the help of DNA sequencing.

(%I DNA

Plasmids 32°C 42°C fragments ACKNOWLEDGMENTS

We thank Dr. E. Lederberg for plasmids and Dr. pNK7 7.3 30.9 a, b, g Tatsuo Yamamoto for the fruitful discussion. Thanks pNK28 0.8 3.1 g are also due to Dr. Sheldon Finver, Mr. Jack Milligan, pNK33 0.0 0.0 a, g and Mr. Robert Ricker for helpful suggestions and advice pNK98 0.0 1.0 a’ on preparation of the manuscript. This work was partly pNKlO0 0.0 0.3 supported by Grant PHS GM- 12053. pNKl08 0.5 30.3 t pNKll1 7.5 74.6 b’ pNKl16 0.8 4.2 b> g

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Documents

Identification of an Rts1 DNA fragment conferring temperature-dependent instability to vector plasmids