10.1101/SQB.1983.047.01.076Access the most recent version at doi: 1983 47: 647-653Cold Spring Harb Symp Quant Biol

 S. Hattman, M. Goradia, C. Monaghan, et al. Bacteriophage MuRegulation of the DNA-modification Function of  

References

http://symposium.cshlp.org/content/47/647#related-urlsArticle cited in:  

http://symposium.cshlp.org/content/47/647.refs.htmlThis article cites 27 articles, 10 of which can be accessed free at:

serviceEmail alerting

click herethe box at the top right corner of the article orReceive free email alerts when new articles cite this article - sign up in

http://symposium.cshlp.org/subscriptions go to: Cold Spring Harbor Symposia on Quantitative BiologyTo subscribe to

Copyright © 1983 Cold Spring Harbor Laboratory Press

Cold Spring Harbor Laboratory Press on May 21, 2012 - Published by symposium.cshlp.orgDownloaded from

Regulation of the DNA-modification Function of Bacteriophage Mu

S. HATTMAN,* M. GORADIA,t C. MONAGHAN,t AND A.I. BUKHARIt *University o f Rochester, Department o f Biology, Rochester, New York 14627; tCold Spring Harbor Laboratory,

Cold Spring Harbor, New York 11724

The DNA-modification function of bacteriophage Mu, termed the mom function, presents very interesting examples of DNA modification and regulation of gene expression. On both counts it sets new precedents. The modification involved is new, and the expression of the mom gene appears to require methylation of sequences adjacent to the gene.

Toussaint (1976) reported that bacteriophage Mu is protected against restriction systems such as that of bacteriophage P I. She also studied mutants (morn-) that were susceptible to restriction. Allet and Bukhari (1975), while mapping Mu DNA by restriction en- zymes, found that Mu DNA made by inducing temperature-inducible lysogens could not be digested with the enzyme HindlI , but was cut normally by EcoRI

and HindlII . In contrast, when the phage was grown by infection, the DNA could be cleaved by HindlI . This correlated with Toussaint's observation that Mu grown by induction was more protected against some restric- tion systems than Mu grown by infection. Furthermore, Allet and Bukhari (1975) showed that a mom- mutant DNA was digested to completion by HindlI . Thus, it was clear that Mu DNA was chemically modified in a sequence-specific fashion, and this modification re- quired the functioning of the gene mom. R. Kahmann and D. Kamp, at Cold Spring Harbor Laboratory, did an extensive study on the cutting of Mu DNA by various restriction enzymes. From the susceptibility of Mu DNA to different enzymes, they deduced that the se- quence modified by mom had a core 5 ' (C/G)-A-(C/G) 3 ' (Hattman 1980; D. Kamp and R. Kahmann, pers. comm.).

Hattman (1979) showed that morn modification pro- duces an unusual modified adenine, Ax, where about 15% of the adenines are modified. Recent studies in- dicate that Ax is N6-carboxymethyladenine but that it is derived from the more acid-labile residue, N 6- (1-acetamido)-adenine (D. Swinton et al., in prep.).

Modified Mu DNA has a T,,, about 4.7~ lower than that of unmodified DNA (S. Hattman, unpubl.).

The mom gene has been mapped at the right end of the bacteriophage Mu genome (Toussaint et al. 1980). As shown in Figure 1, it is the rightmost gene in Mu. The mom gene is far removed from the early genes of Mu and is preceded by at least one gene, gin, that is not under the control of the Mu repressor. However, the morn gene is not expressed constitutively, since in Mu lysogens, DNA is not modified prior to Mu induction; plasmids that contain only the right end of Mu, in- cluding the morn gene, also are not modified.

Requirements for m o r n Action

The expression of mom has turned out to be a complex process, and the signals for its expression react to at least two different factors: (1) a functioning deoxy- adenosine methylase (dam +) gene of Escher ichia coli and (2) a transacting function(s) from bacteriophage Mu.

dam + r equ i r emen t . Toussaint (1977) showed that when mom§ Mu is grown in dam- host cells (Marinus and Morris 1973), the progeny phage particles are modified to less than 1% of the level as in dam + cells. In studies on insertion mutations in Mu, Khatoon and Bukhari (1978) found that DNA from bacteriophage Mu grown in dam- cells was cut to completion by the en- zyme Ball , whereas each site was cleaved in about 50% of the molecules when the phage was grown in dam + cells. Thus, the morn modification of DNA required the dam + methylation function of E. coli. It was originally thought that the morn protein and the dam protein might interact, perhaps lowering the specificity of dam + methylation. This idea turned out to be incorrect when it was realized that the sequence specificity and nature of the modification imparted by mom were entirely dif-

L R 5 I0 15 20 25 30 I I I I I t ( ) ~ _ _ _

I I I I HindIE Pst I Hind~]I Ps~ I

o< G /3

Figure 1. Position of the mom gene on the bacteriophage Mu genetic map. a, G, and/3 are three segments of Mu DNA. Broken lines in- dicate flanking host sequences (Daniell et al. 1973, 1975; Bukhari and Taylor 1975; Bukhari et al. 1976). The room gene is located at the right end (R) in the/3 segment. A repressor gene is located at the left end (L) between the end and the HindlII site. The invertible G segment (indicated by parentheses) encodes proteins needed for Mu adsorption to various hosts. Host range depends on the G orientation (Kamp et al. 1978; van de Putte et al. 1980). The gin product is required for flip-flop of G. In gin- mutants, G inversion is blocked and the phage can be plated only on one type of host (e.g., the G[ - ] or flop orientation does not allow plating on E. coli). The G segment is 3 kb and the/3 segment is 1.6 kb in length. (Segments are not drawn to scale.) Mini-Mu (internally deleted Mu) was made by deleting the middle 28-kb PstI fragment of Mu (Chaconas et al. 1981).

647

Cold Spring Harbor Laboratory Press on May 21, 2012 - Published by symposium.cshlp.orgDownloaded from

648 HATTMAN ET AL.

ferent than for dam + methylation and that morn did not alter the functioning or specificity of dam (Hattman 1979, 1980). This left two reasonable possibilities: (1) only adenine-methylated substrates can be modified by m o m or (2) methylation of DNA is required for expres- sion of morn. The latter possibility is strongly supported by the experiments reported here and by Kahmann (this volume).

Transac t ing f u n c t i o n . Chaconas et al. (1981) con- structed several plasmids containing the right end of Mu and examined their ability to express mom; they ob- served that these plasmids were not able to express the morn gene in the absence of Mu. However, when a mom- Mu prophage was induced in the presence of these plasmids, the DNA was modified. This indicated that the induced mom- prophage was providing a transacting function for expression of the mom gene in the plasmids.

The dam § Function Is Required for Transcription of the morn Gene

If dam* function is required for expression of the mom

gene, then this control may be exerted at the level of transcription. To test this idea it was essential to have a probe for mom-spec i f i c mRNA. Various recombinant plasmids have been produced containing a cloned Mu morn gene. Figure 2 shows a physical map of one such plasmid, pDKXXV-3 (Kwoh et al. 1980), from which the mom gene can be excised on a 1.4-kb PvuI fragment (along with some bacterial DNA). Following digestion with PvuI and agarose gel electrophoresis, this fragment was purified and nick-translated (for a detailed descrip-

tion of methods, see Hattman 1982b). In addition, a total Mu genomic probe was separately prepared by nick translation of virion DNA.

RNAs isolated from uninduced and prophage-induced dam + and dam- E. coli lysogens were electrophoresed through agarose gels, blot-transferred to nitrocellulose filters, and hybridized with 32p-labeled probes. As shown in Figure 3, the 32p-labeled mom probe hybrid- ized only with RNA from the induced dam* Mu lysogen. The low level of m om mRNA in uninduced dam* and dam- lysogens is consistent with poor m om expression in the absence of prophage induction (Toussaint 1976; Toussaint et al. 1980).

Although bacteriophage Mu production is normal in dam- hosts, it was necessary to verify the presence of other Mu-specific mRNAs in these gels. In a parallel analysis, it was shown that the 32p-labeled total Mu probe hybridized equally well with RNAs from induced dam* and dam- lysogens (Fig. 4). Under the hybridiza- tion conditions, little or no Mu mRNA was detected in the uninduced cells. These results are consistent with the fact that Mu development is apparently normal in a dam- host, and they preclude the possibility of extensive degradation of Mu mRNAs.

To rule out the trivial possibility of differential mRNA transfer in the two blots, the same filter was used for sequential hybridization with 32p-labeled mom

and 32p-labeled total Mu probes. In Figure 5 (left), it is

origin --~

I Uninduced Induced

dam + Idarn - dam +1 dam -

Figure 2. Schematic diagram of the physical map of plasmid pDKXXV-3. (,~,~r Bacterial DNA; ( ~ ) Mu genes; ( . ) plasmid DNA. Outer arrows represent distances between restric- tion sites for Hindll (D. Kwoh, pers. comm.), and arrows pointing to closed circles indicate PvuI sites (S. Hanman, unpubl.). Nucleotide sequence data reveal, in fact, that there are two very closely spaced Pvul sites within the Mu DNA (Kahmann, this volume). From the Mu DNA sequence, we calculate that the 1.4-kb Pvul fragment contains 1.0 kb of Mu DNA and 0.4 kb of bacterial DNA.

m o m - , ~

Figure 3. Hybridization of the 3:P-labeled morn probe to RNA from uninduced and induced Mu lysogens (Hattman 1982b). For each RNA sample, 4-, 8-, and 16-#g aliquots were subjected to electrophoresis.

Cold Spring Harbor Laboratory Press on May 21, 2012 - Published by symposium.cshlp.orgDownloaded from

REGULATION OF THE MU MOM GENE 649

Figure 4. Hybridization of the s2p-labeled total Mu probe to RNA from uninduced and induced Mu lysogens.

again evident that morn mRNA is present at a much lower concentration in the induced d a m - lysogen; it is estimated that the d a m + host contained at least 20-fold more morn RNA than the dam- strain. In the secondary hybridization with the a2p-labeled total Mu probe, both d a m + and d a m - cells exhibited similar patterns of hybridization (Fig. 5, right). In this experiment, a reduced amount of total Mu probe was used and, conse- quently, one can discern distinct mRNA bands. It is also evident that morn mRNA is smaller than the bulk of the prevalent Mu RNA species present at 30 minutes postin- duction. To obtain an estimate of the size of morn

mRNA, replicate RNA samples were fractionated by electrophoresis through agarose; the gel was sliced longitudinally, and one half of the gel was stained with ethidium bromide to reveal 16S and 23S ribosomal RNAs. The other half of the gel was blotted and hybridized with the 32p-labeled morn probe. From a comparison of the migration distances, it is estimated that morn mRNA is about 1100 nucleotides long (data not shown); however, further work is required to establish this more precisely.

The foregoing provides evidence that transcription of the Mu room gene is dependent on the host d a m +

methylase activity. There are two possible modes in which the methylase might exert its regulatory function: (1) by virtue of its methylating activity or (2) by binding to a specific site. In the latter mode the enzyme would have a function like the E. col i catabolic activator pro- tein (CAP). We favor methylation per se for the fol- lowing reason: A functional bacteriophage T4 DNA- adenine methylase gene has been cloned into plasmid pBR322 (S. Schlagman and S. Hattman, in prep.). After transferring the plasmid into a dam- host, we observed that the T4 methylase was capable of promoting Mu room § modification. We expect that the bacteriophage T4 enzyme is much different than the E. co l i d a m +

methylase (although both enzymes are capable of methylating GATC) (Brooks 1977; Lacks and Green- berg 1977; Hattman et al. 1978). This follows from the fact that the E. col i d a m + gene codes for a polypeptide with a molecular weight of 31,000 (Herman and Mod- rich 1982), compared with only about 15,000 for T2 d a m + (and presumably T4 d a m +) (Brooks 1977). Moreover, under stringent conditions, there is no ap- parent homology between the E. col i and T4 d a m genes (S. Schlagman and S. Hattman, in prep.; J.E. Brooks, pers. comm.). Thus, it seems likely that the T4 and E. col i enzymes act by virtue of their ability to methylate GATC sequences, rather than by their sharing a com- mon binding site on Mu DNA. Therefore, for the re- mainder of this paper, we will assume that d a m +

methylation has a positive regulatory role in m o m - g e n e

transcription.

Figure 5. Sequential hybridization of a2p-labeled probes to RNA from induced Mu lysogens. (Left) Primary hybridization was car- ried out with the 32p-labeled morn probe. (Right) After autoradio- graphy, the filter was used for a secondary hybridization with the 32p-labeled total Mu probe.

The dam Function Acts at Sites Upstream from the m o m Gene

The requirement for d a m + methylation for transcrip- tion implies that there is methylation of sequences

Cold Spring Harbor Laboratory Press on May 21, 2012 - Published by symposium.cshlp.orgDownloaded from

650 HATTMAN ET AL.

located adjacent to, or overlapping, sequences required for synthesis of morn mRNA. To test whether manipula- tion of the DNA adjacent to the morn gene would change the requirements for its expression, we deleted se- quences in the vicinity of the mom gene. This was car- ried out by opening the plasmid pGC501 (Chaconas et al. 1981) at the PstI site and then degrading the DNA with the BAL-31 exonuclease. This generation of dele- tions is illustrated in Figure 6. As shown, pGC501 con- tains the right end (1.6 kb) and the left end (1.6 kb) of bacteriophage Mu, separated by a gene for ampicillin resistance. After excision of the small PstI fragment, digestion with the BAL-31 exonuclease produced pro-

Hind TTr P~tl I ,PstI Hind m \ ~ ~ n XhoI~/ ~Pvu~

Hind "fl'r ~ ~

Pst T

gressively longer deletions, moving clockwise and counterclockwise from the two PstI ends, respectively. The deletion plasmids generated in this fashion were assayed for their Gin and Mom phenotypes. They formed three classes: gin* mom § gin- morn § and gin-

mom-. The mom§ plasmids were transferred to a dam- strain containing a Mu morn- prophage (Bu2227), and the expression of mom was examined. The results are summarized in Table 1. Plasmids pGC501 (an amp'

mini-Mu plasmid), pGC302 (containing the left end of Mu), pCL151 (containing the right end of Mu), and pGC121 (a mini-Mu lacking the middle 28-kb PstI frag- ment of Mu; see Fig. 1) were described previously (Chaconas et al. 1981). Plasmid pGC121 showed about 50% modification of a morn- prophage induced in a dam § host. However, in dam- cells, the modification was 100-fold to 1000-fold lower. In pGC501, the plasmid from which the deletions were derived, the dam dependence is reduced, and the modification is about 30-fold lower in dam- cells. This probably reflects the insertion of a gene for amp r at the extreme right end of the G segment (Leach and Symonds 1979); i.e., it may be because of readthrough from the/3-1actamase (amp r)

gene. Plasmid pGC302, which lacks the morn gene, showed no modificatin in dam- or dam+ cells. In con- trast, with plasmid pCL151 containing the mom gene,

Hind n7 XhoT I I

PvuF 1 morn gin

c S

~ Bal 31

morn gin

blunt end ,,~ igote

HindTn" �9 ~.~

Q) F i g u r e 6. Strategy used in p roduc ing in vitro deletion mutants o f pGC501 . The min i -Mu plasmid, pGC501 (20 #g) (Chaconas et al. 1981), was l inearized by digest ion with 20 units o f Pstl . The D N A was then d iges ted with BAL-31 exonuclease (1 unit /~g DNA) at 2 0 ~ in a react ion volume of 50 #1. Aliquots were removed at 5, 10, and 20 min, and the react ion was stopped on ice by the addit ion o f E D T A to 50 raM. The D N A was then made b lun t -ended by repa i r synthes i s wi th the K l e n o w f r agmen t o f E. col i D N A polymerase , and the blunt-ended molecules were c i rcular ized with T4 DNA ligase. The reaction mixture was used to t ransform strain 610, and te tracycl ine-resis tant colonies were selected.

T a b l e 1, Effect o f Deletions on morn Funct ion

Modification index

Gin Mu morn- induction Mu morn- induction Plasmid phenotype in a dam" host in a dam- host

None 3 x 10 -4 1 x 10 -4 pCG121 + 6 x 10- ' 2 . 9 x 10- ' pGC501 + 8 x 10 - ' 2 .6 x 10 -2 pCL151 + 7 x 10 -3 3 . 0 x 10 -4 pGC302 - 1.3 x 10 -4 2 .0 x 10- ' A28 + 2 x 10 -~ 2 x 10- ' A l L - 1 x 10 -* 7 x l0 -s A1Q - 8 x 10 -~ 2 . 3 x 10 -2 A29 -- 7 X 10 -2 1 X 10 -t A41 -- 4 X 10 -2 3 . 5 X 10 -t A49 -- 2 X 10 -t 5.1 X 10 -1 a 5 0 - 5 X 10 -~ 6i7 X 10 -2 A69 - 1 • 10 -t 1.3 x 10- ' A l I - 4 X 1 0 - I 1 . 2 X 10 -1 A1N - - 7 X 10 -1 1,5 X 10- '

Modification of bacteriophage Mu morn- induced in the presence of various deletion plasmids. The deletion plasmids were transformed into strain Bu165, which is a dam" lysogen of Mucts62 morn3452 (Toussaint 1976), and Bu2227, which is a dam-4 (GM124 of M. Marinus, University of Massachusetts Medical School) lysogen of Mucts62 morn3452 phage. Lysogen cultures (10 ml) were grown with aeration at 32~ to a density of about 5 x 108/ml. They were then transferred to 42"C and incubated until lysis. The supernatant was plated on strain 610 and a PI lysogen of 610 (Bu2217). An index of morn modification is the ratio of the number of plaques on a PI" lysogen to the number of plaques on a PI- strain. Thus, the closer the ratio is to unity the greater the extent of morn-modification expres- sion. To score for the Gin- phenotype, the plasmids were transferred to strain Bu7045, which contains the gin- prophage 445-5 (Chow et al. 1977). In this prophage, the G segment of Mu remains in the negative orientation such that the progeny phage are not infective for E. coil (Kamp et al. 1978). If gin" function is provided, the G segment is inverted and the phage pro- duces the appropriate proteins that allow progeny phage to form plaques on 1s coli.

Cold Spring Harbor Laboratory Press on May 21, 2012 - Published by symposium.cshlp.orgDownloaded from

REGULATION OF THE MU MOM GENE 651

there was some modification that was d a m * - d e p e n d en t .

We consistently obtained lower modification of Mu DNA when only the right end of Mu was present on the plasmid, as compared with mini-Mu, which contained both the left and right ends. The deletion plasmids showing the Gin* Mom§ phenotype also showed a dam*-dependent m o m function (0.01-0.001 of the m o m

modification index in d a m - cells, as compared with dam* cells). An example is the deletion strain 28. However, the gin- morn § plasmids showed various levels of activities in the presence and absence of dam*.

Although in some plasmids the morn gene was still fully dependent on dam* (e.g., ALL), in others the morn func- tion has become completely independent of d a m .

Plasmids 41 and 49 showed the highest level of morn ac- tivity in d a m - bacteria observed so far. These results show that altering the structure of DNA adjacent to the morn gene can have profound effects on the functioning of the gene. One of the effects is that the expression of the gene is no longer dependent on dam+ methylation.

We also tested a well-characterized insertion-deletion mutant of bacteriophage Mu for its Morn phenotype. One such insertion was isolated in pGC 121 as a gin- mu- tant and has no gross deletion (R. Harshey, pers. comm.). Plasmid pRA6 (see Fig. 7) was found to be phenotypically Morn-, since it did not complement a Mu m o m - prophage. The plasmid was transferred to a rho-

strain (lacking the transcription termination factor rho) .

In rho- strains, transcription proceeds through the rho-

dependent terminators, frequently relieving the polarity of insertion mutations, pRA6 was found to be pheno- typically Mom* in a rho- background (Table 2). This result shows that the m o m gene is intact in pRA6. Since insertion in g in has eliminated the expression of the morn gene, this also shows that transcription of morn is from left to right in the Mu genetic map (Fig. 1). Fur- thermore, this raises the possibility that the expression signals of morn overlap the g in gene. Expression of the morn gene in the rho- background most likely occurs from a promoter in the insertion sequence, which has not been identified so far.

We also tested a well-characterized insertion-deletion mutant of bacteriophage Mu for its Morn phenotype. This mutant, 445-5, has been described by Chow et al. (1977) and Chow and Broker (1978). It has a deletion in the g i n gene and a remnant of the IS2 element. This

IS c7 \/

G )l gin I morn ~ . ~

Pst T~- - pRA6 Figure 7. Structure of a plasmid containing an insertion in the gin gene. The plasmid is a gin- derivative of pGC 121 (Chaconas et al. 1981). ( ) Plasmid pSCI01; ( ) Mu DNA from the right end of the phage genome (see Fig. 1). The G segment is indicated by parentheses. IS indicates the insertion (the precise location has not yet been identified). Segments are not drawn to scale.

Table 2. rnom Expression in rho- Background

Strain Modification

p R A 6 / M u m o m - / r h o § (1338) 5 x 10 -4 pRA6 /Mumom- / rho - (1336) 1.7 x 10 -1

Mucts62, 445-5/rho § (1338) 2 x 10 -s Mucts62, 445-5/rho- (1330) 1 x 10 -2

Mucts62 mom§ § (1338) 6 x 10 -1 Mucts62 mom§ - (1336) 2.7 • 10-'

Mucts62 mom-/rho* (1338) 1.3 • 10 -6 Mucts62 mom-/rho- (1336) 1.4• 10 -s

For procedure and modification index, see Table 1. The strains were Bu1336 rho*, lacU169 and Bu1338, rho-201, lacU169 (obtained from J. Beckwith, Harvard Medical School) and Bu1330, rhotsl5, his-gal-3 val r (obtained from A. Das, University of Connecticut Medical School).

substitution was derived from an IS5-IS2 tandem inser- tion in the middle of the/3 region of bacteriophage Mu. We again found that the structural gene for m o m is intact in the mutant, since the bacteriophage becomes Morn § in a rho- background (Table 2). Presumably, m o m tran- scription is being initiated from within the IS2 sequences.

From the results on the direction of m o m transcrip- tion, and on the effects of the BAL-31 deletions, we conclude that dam* methylation of sequences upstream from the morn gene controls the transcription of m o m .

The Transactivation Requirement Can Be Overcome

Since the modification imparted by the m o m gene is complex, it is possible that the so-called transacting function is an accessory protein required for the chemical modification of the adenine residues. Alter- natively, it may be required mainly for the expression of the m o m gene; i.e., the transacting function may play a role in initiation of transcription or in antitermination of transcription. We tested some of the plasmids whose function is independent of d a m to determine whether they expressed the m o m function in the absence of a Mu prophage. In these plasmids the left end of Mu confin- ing the c gene (the repressor gene) is joined to the morn

gene. This fragment contains a rightward promoter that overlaps the Hind l I I site (Priess et al. 1982). If the m o m

gene is hooked onto this promoter, it may be repressed by the Mu repressor, since this region defines the im- munity region of Mu. We therefore grew the strains at 32~ and 42~ and made plasmid preparations. Since the plasmids encode a temperature-sensitive Mu repres- sor, it would become inactive at 42~ allowing the ex- pression of the m o m gene if it were properly joined to the immunity region of Mu. Initially, we transformed P1 lysogens (the restricting hosts) and a P1- strain (a nonrestricting host) to monitor modification of the plasmid DNA. The number of transformants obtained in a P1 lysogen, compared with that obtained in a P1- strain, would be indicative of plasmid modification. There was no significant difference in the number of

Cold Spring Harbor Laboratory Press on May 21, 2012 - Published by symposium.cshlp.orgDownloaded from

652 HATTMAN ET AL.

transformants obtained. Therefore, none of the plasmids were fully modified at either a low or high temperature. However, when we cut the plasmid with the enzyme HgaI (whose recognition sequence GACGC is modified by mom) , it was apparent that the plasmid was partially modified at high temperature, but not at low tempera- ture (Fig. 8), There may be more modification at high temperature in a rho- background; however, the results are too preliminary to reach a firm conclusion at this time. Since no bacteriophage Mu is present in these strains, these results imply that when morn is linked to a different promoter, the requirement for transactivation can be overcome. Finally, when the mom gene was in- serted into the amp ' gene of pBR322, we obtained no viable clones when the m o m - g e n e orientation was aligned with the ampr promoter (S. Hattman and J. Ives, in prep.). This suggests that these clones expressed m o m modification constitutively (a lethal event) and in the absence of transactivation. We conclude that the transacting function is involved in activating transcrip- tion of the m o m gene.

GENERAL DISCUSSION

The methylation-dependent functioning of the m o m

gene is the first clear-cut example of the regulation of

Hga r

mod i f ied rho- rho §

r - - l r - - 1 uncut

Figure 8. Expression of mom without transactivation by Mu. A rho* strain (Bu1338) and a rho- strain (Bu1336) and a Mu lysogen (HM8305) were transformed with deletion plasmid 49 (see Table 1). The rho § and rho- derivatives were grown at either 32~ or 42~ The HM8305 derivative was grown to a density of 108 cells/ml at 32~ and then transferred to 42~ for 1 hr. Small-scale plasmid preparations were made from these cultures and digested with Hgal. Digests were run on a 0.7% agarose gel with 0.5 #g/ml of ethidium bromide and visualized under UV light. The bright fluorescence at the bottom of the gel is due to the presence of RNA in these crude plasmid preparations.

gene expression by methylation. In eukaryotes, methylation of cytosine residues has been proposed to turn off gene expression, and the idea is gaining strength that certain active regions of chromosomes are inac- tivated by methylation (for review, see Razin and Fried- man 1981; Wigler 1981; and Hattman 1982a). In the case of the morn gene, however, methylation seems to have a positive regulatory role in turning on the morn

gene. Perhaps there are other prokaryotic genes that are positively (or negatively) regulated by dam" or dcm+

(deoxycytidine methylation). The fact that DNA- methylation-defective mutants are viable and propagate phage suggests that no essential host or viral gene is positively regulated. Nevertheless, it would be worth- while to screen nonessential genes for such a regulatory control.

What is the mechanism by which the m o m gene is turned on? We have presented evidence that transcrip- tion of the m o m gene requires an active dam* function of the host and that this requirement for dam* can be cir- cumvented by deleting DNA sequences upstream from the m o m gene. The latter is in agreement with the find- ings of Kahmann (this volume). Apparently, dam*

methylation of a specific region is required for trans- cription (this region appears to be the block-one se- quence described by Kahmann [this volume]). Whether dam § methylation is required for initiation of transcrip- tion or for preventing termination of nascent mRNA is not clear. It does seem that the regulatory sequences of m o m overlap the gin gene, and alterations in this region have a dramatic effect on the dam* requirement for m o m - g e n e expression. Under normal circumstances, dam* methylation is a necessary, but not sufficient, con- dition for mom-gene expression. This follows from the observation that m o m modification is not constitutively expressed by plasmids containing a cloned m o m gene; however, infection with the m o m - phage results in trans- activation of the m o m gene present on the plasmid. It would seem that the transacting protein (TAP) recognizes the presence or absence of methylation, and it is the critical regulatory element that allows RNA polymerase to transcribe mom.

In addition to the dam* methylase and TAP re- quirements, it should be noted that prophage induction is essential to obtain efficient m o m modification (Tous- saint 1976); i.e., during lytic infection, modification is incomplete. However, recent studies show that there are exceptions to this. For example, efficient modification is observed following lytic infection of cells containing certain plasmids with a cloned m o m § gene (Chaconas et al. 1981; S. Hattman, unpubl.) or a cloned E. coli or T4 dam § methylase gene (S. Hattman, unpubl.). At present, the reason for the different modification efficiencies elicited by lytic infection versus prophage induction is not understood. It is possible that the difference arises from the different levels of methylation in phage DNA (where not all molecules may be fully methylated) ver- sus prophage DNA (which is fully methylated).

As mentioned above, the Mu system presents an ex- traordinary case of DNA modification and the regula-

Cold Spring Harbor Laboratory Press on May 21, 2012 - Published by symposium.cshlp.orgDownloaded from

R E G U L A T I O N OF T H E M U MOM G E N E 653

tion o f gene expression through D N A modificat ion. Although we have gained a considerable understanding of these processes in the last few years, it is evident that detailed mechanisms remain to be elucidated.

A C K N O W L E D G M E N T S

This work was supported by a U.S. Public Health Ser- vice grant (GM-29227) to S.H. and grants from the Na- tional Science Foundation and the National Institutes of Health to A.I .B.

R E F E R E N C E S

ALLET, B. and A.I. BUKHARI. 1975. Analysis of bacter- iophage Mu and iambda-Mu hybrid DNAs by specific en- donucleases. J. Mol. Biol. 92: 529.

BROOKS, J.E. 1977. " A comparative study of the wild-type and mutant forms of phage T2 DNA-adenine methylase." Ph.D. thesis, University of Rochester, Rochester, New York.

BUKHAR1, A.I. and A.L. TAYLOR. 1975. Influence of inser- tions on packaging of host sequences covalently linked to bacteriophage Mu DNA. Proc. Natl. Acad. Sci. 72: 4399.

BUKHAPa, A.I., S. FROSHAUER, and M. BOTCHAN. 1976. Ends of bacteriophage Mu DNA. Nature 264: 580.

CHACONAS, G., F.J. DE BRUIJN, M.J. CASADABAN, J.R. LuP- SKI, T.J. KWOH, R.M. HARSHLY, M.S. DU BOW, and A.I. BUKHARI. 1981. In vitro and in vivo manipulations of bacteriophage Mu DNA: Cloning of Mu ends and construc- tion of mini-Mu's carrying selectable markers. Gene 13: 37.

CHow, L.T. and T.R. BROKER. 1978. Adjacent insertion se- quences IS2 and IS5 in bacteriophage Mu mutants and an IS5 in a lambda darg bacteriophage. J. Bacteriol. 133: 1427.

CHow, L.T., R. KAHMANN, and D. KAMP. 1977. Electron microscopic characterization of DNAs of non-defective deletion mutants of bacteriophage Mu. J. Mol. Biol. 113: 591.

DANIELL, E., D.E. KOHNE, and J. ABELSON. 1975. Char- acterization of the inhomogeneous DNA in virions of bacteriophage Mu by DNA reannealing kinetics. J. Virol. 15: 739.

DANIELL, E., J. ABEl,SON, J.S. KIM, and N. DAVIDSON. 1973. Heteroduplex structures of bacteriophage Mu DNA. Virology 51: 237.

HATTMAN, S. 1979. Unusual modification of bacteriophage Mu DNA. J. Virol. 32: 468.

- - . 1980. Specificity of the bacteriophage Mu mom*-con- trolled DNA modification. J. Virol. 34: 277.

- - . 1982a. DNA methylation. The Enzymes 14: 517. - - . 1982b. DNA methylase-dependent transcription of the

phage Mu morn gene. Proc. Natl. Acad. Sci. 79: 5518. HATTMAN, S., J.E. BROOKS, and M. MASUREKAR. 1978. Se-

quence specificity of the Pl-modification methylase (M.Eco PI) and the DNA methylase (M.Eco dam) con- trolled by the E. coli dam gene. J. Mol. Biol. 126: 367.

HERMAN, G.E. and P. Moolucm 1982. Escherichia coli dam methylase. Physical and catalytic properties of the homo- geneous enzyme. J. Biol. Chem. 257: 2605.

KAMP, D., R. KAHMANN, D. ZIPSER, T.R. BROKER, and L.T. CHOW. 1978. Inversion of the G segment of phage Mu con- trols phage infectivity. Nature 271: 577.

KHATOON, H. and A.I. BUKHARI. 1978. Bacteriophage Mu- induced modification of DNA is dependent upon a host function. J. Bacteriol. 136: 423.

KWOH, D.Y., D. ZIPSER, and D.S. ERDMANN. 1980. Genetic analysis of the cloned genome of phage Mu. Virology 101: 419.

LACKS, S. and B. GREENBERG. 1977. Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. J. Mol. Biol. 114: 153.

LEACH, D. and W. SYMONDS. 1979. The isolation and characterization of a plaque-forming derivative of bacteriophage Mu carrying a fragment of Tn3 conferring ampicillin resistance. Mol. Gen. Genet. 172: 179.

MAPaNUS, M.G. and N.R. MORRIS. 1973. Isolation of deoxy- ribonucleic acid methylase mutants of Escherichia coli K-12. J. Bacterol. 114: 1143.

PR1ESS, H., D. KAMP, R. KAHMANN, B. BRAUER, and H. DELIUS. 1982. Nucleotide sequence of the immunity region of bacteriophage Mu. Mol. Gen. Genet. 186: 315.

RAZIN, A. and J. FreEDMAN. 1981. DNA methylation and its possible biological roles. Prog. Nucleic Acid Res. Mol. Biol. 25: 33.

TOUSSAXNT, A. 1976. The DNA modification function of temperate phage Mu-l. Virology 70: 17.

- - . 1977. DNA modification of bacteriophage Mu-1 re- quires both host and bacteriophage functions. J. Virol. 23: 825.

TOUSSAINT, A., L. DESMET, and M. FALLEN. 1980. Mapping of the modification function of temperate phage Mu-l. Mol. Gen. Genet. 177: 351.

WIGLER, M.H. 1981. The inheritance of methylation patterns in vertebrates. Cell 24: 285.

VAN DE PUTTE, P., S. CRAMER, and M. G1PHART-GASSLER. 1980. Invertible DNA determines host specificity of bacteriophage Mu. Nature 286:218.

Cold Spring Harbor Laboratory Press on May 21, 2012 - Published by symposium.cshlp.orgDownloaded from