Effects of DNA heterologies on bacteriophage lambda packaging.

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Copyright 0 1988 by the Genetics Society of America

Effects of

DNA

Heterologies on Bacteriophage

X

Packaging

Rebecca

Kurth

Pearson’ and Maurice S. Fox

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Manuscript received April 3, 1987 Revised copy accepted September 18, 1987

ABSTRACT

We have examined the impact of D N A heterologies on the packaging of X DNA in vitro. Heterology- containing D N A molecules were constructed by denaturing and reannealing a mixture of D N A from

cI+ phage and D N A from phage carrying small insertion or deletion mutations in the cI gene. We found that molecules with heterologies of up to 19 base pairs (bp) can be packaged as viable heterozygous phage with approximately the same efficiency as molecules with a base pair mismatch. In contrast, with a heterology of 26-bp heterozygous plaque formers are rare. I n principle, the absence of cI heterozygotes among packaged phage may be due either to a failure to encapsidate the DNA or a failure to inject the packaged DNA on infection. Southern blot analysis of DNA isolated from packaged phage indicates that DNA harboring a 26-bp heterology is almost completely absent in packaged phage. Thus, an upper limit has been established for the size of heterology that can be accommodated by the packaging apparatus. The size of the connector portal could be the basis for this limit.

P

RODUCTS of recombination harbor regions of

heteroduplex

D N A

(for review, see FOX 1978;

RADDING 1982). If recombining parental molecules

are distinguished by the presence of an insertion or deletion allele in one of them, the inclusion of such an allele within the heteroduplex region would result in the presence of a heterology. In the case of bacteriophage, the presence of such structures could com- promise the encapsidation process and thereby influ- ence the likelihood of recovering them as viable recombination products. Several investigations of phage

T4 morphogenesis have suggested that the encapsi-

dation or “packaging” of

D N A

by bacteriophages is in fact sensitive to structural alterations in the

D N A

duplex (for review, see BLACK and SHOWE 1983).

T h e bacteriophages which contain doub1e;stranded

D N A

are thought to package

D N A

by the same fun- damental mechanism, and many aspects of the process

have been characterized both genetically and bio-

chemically for several phages (for review, see BAZINET

and KING 1985; EARNSHAW and CASJENS 1980).

Three basic components interact during packaging: concatemeric

D N A ,

a processed precapsid or “prohead,” and certain auxiliary packaging proteins. T h e

D N A

and proheads are the products of two distinct synthetic pathways of the phage. T h e

D N A

replication

pathway produces concatemers of phage chromo-

somes, while coat proteins are assembled into pro-

heads in the head morphogenetic pathway. These two

pathways converge in the packaging reaction, when concatemeric

D N A

is linked to the unique “portal”

’

Present address: Department of Cell Biology, Biocenter of the Univer- sity, Klingelbergstrasse 70, Basel, Switzerland.

Genetics 1 1 8 5-12 (January, 1988)

vertex of proheads through the action of special pack-

aging proteins which also mediate the subsequent

encapsidation process. Neither the energetic basis nor

the physical mechanism underlying

D N A

transloca-

tion and condensation into the prohead is understood for any bacteriophage.

Many mutations have been characterized which re-

sult in prevention of

D N A

packaging, and most of these are characterized by an alteration of the structure of the prohead or auxiliary packaging proteins.

Although certain

D N A

structural alterations are

known to prevent packaging in phage T 4 (KEMPER

and JANZ 1976; HSIAO and BLACK 1978; ZACHARY

and BLACK 1981, 1984), the structural requirements

of the

D N A

substrate for packaging remain poorly defined. In particular, it is not known what effect

D N A

heterologies have on the process of

D N A

encap-

sidation. Since encapsidation seems to require

D N A

passage through the portal vertex, investigation of the effect of the presence of

D N A

heterologies on this

process could reveal some features of the tolerance of

the packaging mechanism to perturbations in the duplex

D N A

structure.

T h e effect of

D N A

heterologies on h packaging in vitro has been examined. The heterologies were created by denaturing and reannealing

D N A

from c l +

phage with

D N A

from phage carrying small insertion or deletion mutations in the c l gene. We show that heterologies of up to 19 bp can be packaged in vitro

with approximately the same efficiency as a base pair

mismatch; phage heterozygous for c l are found among

the viable products of phage packaging.

A

heterology

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TABLE 1

Bacterial strains

Designation Relevant genotype

FA77 dnaBts, su-

M182 lacZ galU galK rpsL

M182mutL mutL218::TnlO lac2 galU

galK rpsL

C600 thr leu lacy sull

C6OOmutL mutL218::TnlO thr leu lacy

S U I 1

BNN45 met gal lacy r-m+ sull suII1

Source

MCMILIN and Russo ( 1 970)

E. SIGNER

S. RAPOSA

APPLEYARD HUISMAN and FOX

(1 986)

D. BOTSTEIN

TABLE 2

Phage strains

Genotype Source

P a m 8 0

Ac160 Oam29

A c I L W ? Pam80 Sam7 plac5ZamNG200 AOam29

Ad286 Oam29 Ad287 Oam29 Ad288 Oam29 Ad289 Oam29 Ad290 Oam29 Ad291 Oam29 kc1292 Oam29 AclAH? Oam29

E. SIGNER

LICHTEN and Fox (1 983)

M. LICHTEN

PEARSON and Fox (1 988)

PEARSON and Fox (1988)

PEARSON and Fox ( 1 988) PEARSON and Fox ( 1 988) PEARSON and Fox (1 988)

a 10-fold or more reduction in t h e yield of c l heterozygotes. Their frequency is near the limit of t h e detection methods used. In principle, this absence of

c l heterozygotes among the packaged phage may be

due either to a failure to encapsidate the DNA or a failure to inject t h e DNA from the phage packaged with this DNA into a bacterial host. Restriction analysis of DNA isolated from packaged phage indicates that DNA harboring the 26-bp heterology is excluded from packaging. These results suggest that it is some aspect of t h e X packaging process, rather than the injection process, that is sensitive to the size of DNA heterologies.

MATERIALS AND METHODS

Bacterial strains and phage strains: Bacterial and phage

strains are described in Tables 1 and 2, respectively.

Media and buffers: Media for growing cultures, diluting

and plating phage have been described by PEARSON and Fox (1 988).

Preparation of artificial heteroduplex and sham DNA:

High titer phage stocks were grown, concentrated, and purified on density step gradients as described by LICHTEN and Fox (1983). An ODnso measurement of the dialyzed phage preparation was used to estimate its DNA concentration. High titer phage were diluted into SM buffer containing 20 mM EDTA and mixed at a concentration of 50 pg DNA/ml of each parent (1 00 pg/ml total). After "popping" the phage by heating at 65" for 15 min, the phage DNA

mixture was divided into two aliquots. One half was used to prepare heteroduplex (het) DNA: the sample was denatured by adding 1/10 volume 1 M NaOH. After 20 min at room temperature, the mixture was neutralized with 1/10 volume of 2 M Tris-HCI (pH 7.2). The second aliquot of phage DNA was used to make "sham" control DNA: The NaOH and Tris-HCI solutions described above were added simul- taneously to the DNA and allowed to stand at room temperature for 20 min. An equal volume of formamide was added to both het and sham samples and the mixtures were incubated for 1 hr at 37". Small samples of the het and sham DNAs were then diluted 1/100 into 20 mM Tris-HCI (pH 8.0) to be used for transfections. The remaining het and sham DNA mixtures were dialyzed for 48 hr at 4" against 1 liter of STE buffer (10 mM Tris-HCI, pH 8.0, 1

mM EDTA, 100 mM NaCI) with three changes, then for 12 hr against 1 liter of 2.5 mM Tris-HCI, 0.1 mM EDTA (pH 8.0). The dialyzed het and sham DNA samples were concentrated IO-fold by evaporation in a Speed-vac. To promote concatemerization, the DNA samples made up to 10 mM

MgClp were incubated at 42" for 2 hr.

Transfection: One hundred microliters of het or sham

DNA at approximately 0.5 pg/ml was heated 10 min at 65 O

to monomerize the DNA and then quickly chilled on ice. Each sample was mixed with 200 pl of ice-cold calcium- treated competent CGOOmutL cells (MANDEL and HIGA 1970) and successively incubated 15 min on ice and 30 min at 37". Infective centers were plated on trypticase plates, along with 0.2 ml of a fresh plating culture of BNN45 and 2 ml of X top agar. Plates were incubated overnight at 37". Twenty microliters of the transfection mixture generally gave 10 to 50 plaques per plate.

Zn vitro packaging: Five microliters of het or sham D N A

were mixed with a thawed Packagene extract (Promega Biotec) and incubated 2 hr at 22". The Packagene extract is prepared with Xcos2 lysogens and therefore will not package endogenous DNA (ROSENBERG et al. 1985). The packaging reaction was stopped by addition of 0.5 ml SM+ buffer (5.85 g/liter NaCI, 20 mM Tris-HCI, pH 7.5, 10 mM MgS04) and 25 pl of CHC13. Packaging efficiencies ranged from 1

X 10' to 3 X lo8 phage per microgram of DNA. Het D N A generally gave about a 5-fold lower yield of phage than its sham counterpart. Packaged phage to be analyzed for cI

allele composition were plated as follows: 0.02 to 0.15 ml of an appropriate dilution of phage was mixed with 0.2 ml of a fresh CGOOmutL plating culture and allowed to adsorb for

15 min at 37". Each sample was then mixed with 2 ml of X

top agar containing 0.2 ml of a fresh plating culture of BNN45 and poured on a trypticase agar plate.

Analysis of plaques for cZ allele composition: Plaques

from transfections and packaged phage were analyzed for their cI allele composition by picking random plaques and streaking them on individual agar plates as described by PEARSON and FOX (1 988).

Isolation of DNA from packaging reactions and from

packaged phage DNA: Five microliters of each het and

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Packaging D N A Heterologies in X 7

aged D N A , 0.4 ml of each sham packaging reaction was mixed with 5 PI of the analogous het DNA and treated with

DNAse as above. The packaged phage were purified o n a step gradient after adding 10"' carrier phage (XclAH3 Sum7

plac5ZamNG200) to each DNAse-treated phage sample.

( T h e carrier phage carry a deletion of the fragment used as

probe in Southern analysis.) Each sample received 2.1 ml of SM+ buffer saturated with CsCl and was layered on the bottom of two 7.0 ml steps of 1.6 g/ml and 1.4 g/ml. The layered tube was centrifuged for 2 hr at 24,000 rpm in a

Beckman SW28.1 rotor and the phage band between the 1.6 g/ml and 1.4 g/ml density steps was withdrawn with a

25 gauge needle. CsCl was removed by dialysis overnight at

4" against SM+ buffer. D N A was extracted from the dialyzed phage by the method of MANIATIS, FRITSCH and SAMBROOK (1 982).

Southern blot procedure: D N A was digested with restric-

tion enzymes (New England Biolabs) according to the sup- plier's instructions, using 10- to 20-fold higher enzyme concentrations. The digests fractionated by electrophoresis i n a 1 % agarose gel, using Tris-borate buffer, and trans- ferred to a Biodyne A nylon membrane (Pall), as described i n MANIATIS, FRITSCH and SAMBROOK (1982). The D N A was immobilized on the Biodyne A membrane by UV- irradiation according to CHURCH and GILBERT (1 984). Hy- bridization conditions and radioactive labeling of the 560

bp cZ/rexA fragment (LICHTEN and FOX 1983) were carried

o u t as outlined by MEINKOTH and WAHL (1 984).

RESULTS

In vitro packaging of heterology containing DNA

molecules: Observations reported previously (PEAR-

SON a n d FOX 1988) have shown that DNA harboring heterologies of up to 19 bp can be matured into viable phage progeny and have suggested that chromosomes harboring heterologies larger than 26 bp cannot. In

order to directly investigate the effect that such structural perturbations have on the maturation of phage

DNA, lambda DNAs containing small heterologies in

c l were prepared and packaged in vitro, and the frequency of heterozygotes among the viable products was determined.

Het and sham DNAs were prepared using D N A

from X c l + Pam80 in combination with DNA from each of several different c l - Oam29 phage harboring insertion or deletion mutations of different sizes. Since sham DNAs have not been denatured, they simply contain a mixture of the two parental c l + Pam80 a n d

c l - Oam29 phage DNAs. Het D N A mixtures, on the other hand, have been denatured and reannealed, so

they contain cl' Pam80/cl- Oam29 heteroduplex

DNA molecules in addition to the reannealed parental phage DNAs. T h e various c l insertion and deletion nlutations would be expected to create heterologies of different sizes in the heteroduplex D N A molecules.

T h e relative amounts of heteroduplex and parental phage DNAs present in the various het and sham

DNA preparations were determined experimentally

by transfection of an amber suppressing (sull) bacterial strain deficient in mismatch repair (C6OOmutL). T h e results are displayed in Table 3. As expected,

TABLE 3

Results of transfection with het and sham DNA

Frequency among transfectants

(X 100)

Observed Predicted

allele change (bp) Turbid Clear het" hetb size

c160 het Base substi- 25 33 42 94

sham tution 37 61 1.9 47 51

c1287 het +4 21 39 40 198

sham 34 62 4.1 45 98

cI289 het +14 20 28 52 99

sham 42 56 2.0 49 50

cI290 het +19 16 30 54 108

sham 48 48 4.0 50 50

~ 1 2 9 1 het +26 27 21 52 100

sham 39 55 6.1 48 49

cI286 het -26 15 49 36 202

sham 39 59 2.1 48 97

cIAH3 het -690 29 45 26 99

sham 45 55 0.0 50 47

GI Sequence Sample

Competent C6OOmutL (sull) cells were transfected with het or sham DNA as described in MATERIALS A N D METHODS. Transfections of c1287 and cI286 het and sham DNAs were carried out on one day and the other transfections carried out on another.

a het: heterozygous. Transfections with sham DNA yield a few

heterozygous plaques presumably as a result of double transfectants. Predicted heterozygous products of transfection were calcu- lated using the quadratic formula, assuming random assortment of reannealed strands. Predicted hets = 2AB, for A = a / a

+

b and B = b/a

+

b where a is the frequency of turbid transfectants and b is the frequency of clear transfectants for sham DNA as listed in the table.

transfections with sham DNA produce almost exclu- sively turbid and clear plaques. T h e few mixed plaques that are observed are presumably the consequence of

double transfection. T h e ratio of turbid and clear plaques observed in transfection with sham D N A can

be used to calculate the frequency of heterozygous plaques to be expected for the corresponding het

DNA (assuming random assortment of strands). T h e predicted and observed frequencies of heterozygous transfectants are in reasonable agreement for almost all of the het DNA preparations. One exception appears to be the het DNA made with the 690-bp deletion phage, which gives a yield of heterozygous transfectants that is modestly lower than expected; similar reductions were also observed in other preparations of 690-bp het DNA (data not shown).

Aliquots from each het and sham DNA preparation were added to X packaging extracts, and the packaged phage were plated on the same suppressing, repair-

deficient strain (CGOOmutL) used in the transfection experiments. T h e results are shown in Table 4. T h e frequency of cl heterozygotes among packaged phage is in all cases lower than expected from the transfection results. A 3-fold reduction is observed with het

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TABLE 4

Results of packaging het DNA

Frequency among packaged phage (X 100) allele

r l Sequence change Sample

(bp) Turbid Clear het size

e160 Base substitution 45 42 13 388

e1287 +4 23 68 9.5 158

e1289 + I 4 50 44 5.8 393

e1290 + I 9 48 44 8.2 357

e1291 +26 59 40 0.8 357

e1286 -26 21 79 0.0 197

c I A W -690 51 49 0.0 362

substitution mutation in c l , suggesting that the pres-

ence of base pair mismatches in heteroduplex DNA

may result in reduction of packaging efficiency. I t

should be noted that in every case the heteroduplex

DNA harbors mismatches at 0 and P , in addition to a mismatch or heterology in c l . Het DNAs prepared with c l - Oam29 phage harboring 4-bp, 14-bp or 19- bp insertion mutations yield similar frequencies of packaged c l heterozygotes. The presence of a heterology of 26 bp results in a 10-fold reduction in the yield of c l heterozygotes, while the yield of c l heterozygotes in phage packaged with a 690-bp deletion heterology falls to the limit of this detection method.

Frequencies of heterozygous phage much below 1 0-2

are not readily detected by screening for c l heterozygotes.

Another approach to the detection of heterozygous packaged phage, which allows us to examine a greater range in heterozygote frequencies, exploits the presence of mismatches at 0 and P in the heteroduplex

DNA. Phage harboring amber mutations in 0 or P

cannot replicate in a nonsuppressing bacterial host and therefore do not produce plaques on su- indicator strains. Phage that are heterozygous for each of these mutations cannot replicate either. However, correction of the heterozygous phage to O+P+ by mismatch repair on the transcribed strand can restore growth in the su- indicator. Thus, the capacity to give rise to

0 + P + plaques on an su- indicator is another way to

detect the presence of heterozygous phage among

packaged products. Table 5 presents the results of titration of the packaged phage on an s u - indicator

(M182mutL). In order to correct for differences in

overall efficiency of packaging, the yield on the su- indicator was normalized to the phage titer detected

on a suppressing (sull) host. Phage packaged with het

DNA prepared from 26-bp and 690-bp deletion phage

show detectable differences in the yield o f ' O+P+

phage. We regard the O+P+ plaques seen with phage

packaged with sham DNA and the 690-bp deletion

het DNA as background, since their frequency is

sensitive to the number of phage plated and can

therefore be accounted for as the products of coinci-

TABLE 5

Results of titering packaged phage on su- and s d Z indicators

Titer

allele change (bp) (xu-) ( s u l l ) ratio (X 100)

C l Sequence M 182mut.L CGOOmutL su-/su/l

e160 het Base substit- 1 . 1 X 10' 5.1 X 1 O7 22

sham ution 1.3 X I O 5 1.5 X 10" 0.09

e1289 het + I 4 2.1 X IO7 1.5 X 10" 14

sham 5.8 X 10' 5.6 X 10" 0.10

e1290 het

+

19 1.0 X lo7 8.6 X I O 7 12

sham 4.3 X IO5 5.0 X 10" 0.09

e1291 het +26 9.4 X 10' 1.2 X l o H 0.78

sham 1.8 X 10' 3.3 X 10" 0.05

c I U 3 het -690 3.0 x 104 3.1 X 107 0.10

sham 5.5 X I O 5 1.0 X IO9 0.06

dent infection. DNA harboring the 26-bp insertion

heterology gives rise to O+P+ plaques at a frequency approximately 8-fold higher than background.

Restriction analysis of packaged phage DNA: In

the experiments described above, phage were identi- fied as heterozygous for GI if they produced plaques containing a mixture of clear and turbid phage gen- otypes. T h e failure to recover c l heterozygotes among

phage packaged with DNA containing 26-bp and 690-

bp heterologies could be due either to a failure to

encapsidate DNA harboring these heterologies or a

failure of the packaged phage harboring these heterologies to produce plaques. For example, the presence

of these heterologies in the phage DNA may interfere

either with threading the DNA into the prohead during packaging or with the process of injecting the

DNA into a bacterial host. These two possibilities were distinguished by purifying phage packaged with the

26-bp and 690-bp het DNAs and then carrying out a

restriction analysis of the DNA isolated from the

packaged phage to determine the presence or absence

of the DNA heterologies. DNA from phage packaged

with the 19-bp heterology was included as a positive control. T h e results are shown in Figure 1.

Digests of unpackaged het DNA produce two bands

that hybridize to the cl/rexA probe. Since het DNA is

made by denaturing and reannealing DNA from a

mixture of c l + Pam80 and c l - Oam29 phage, it contains a mixture of reannealed parental and heterodu-

plex DNAs. With the HindIII-XhoI-MspI digestion

scheme, all reannealed parental DNAs produce a sin-

gle 0.56-kb band that hybridizes to the probe (Figure

2). In heteroduplex DNA containing a 19- or 26-bp

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Packaging DNA Heterologies in X 9

I 2 3 4 5

_-

6 7 8 9 1011 12 1 3 1 4 1 5 T

" " . _

I 9 k b -

1.7 kb -C

l Okb-,

+l9 '26 -690 +l9 4 2 6 -690 +l9 '26 -690 *l9 126 -690 +l9 '26 -690

Unpackaged Packaged Packaged Packaged Packaging Het DNA Het DNA Sham DNA Sham DNA Extracts

(overloaded)

FIGURE 1.-Southern blot o f DNA from packaged phage and packaging extracts. Unpackaged het DNA and DNA isolated from packaged phage and packaging extracts was digested with HindlII, Xhol and Mspl and probed with the rexA/cl Hindlll-Hind111 frag- ment. In all of the samples o f packaging extract DNA, the parental band is smeared; this is also evident with DNA isolated from the extract alone (data not shown) and is therefore probably the con- sequence of endogenous DNA present in the extracts.

with a 690-bp single-stranded loop, which migrates to

the position expected of a 1.9-kb fragment. Digestion of the unpackaged het DNA in all three cases thus gives separate heteroduplex and parental bands, as seen in the autoradiogram. These parental and heteroduplex bands also appear at equal intensities, cor- roborating the transfection results in which the het DNAs gave rise to approximately equal numbers of parental and heterozygous transfection products.

Digestion of DNA isolated from phage which were packaged with the 19-bp het DNA yields both a 0.56- kb (parental) band and a 1.7-kb (heceroduplex) band. There is roughly a 10-fold difference in the relative intensities of the parental and heteroduplex bands. This agrees well with the 8% frequency of c l heterozygotes observed among phage packaged with the 19- bp het DNA. DNA isolated from phage packaged with the 26-bp heterology produces a strong 0.56-kb (parental) band, but only a very faint 1.7-kb (heteroduplex) band. No heteroduplex band is visible in the case of the packaged 690-bp het DNA, but this obser-

vation is compromised by the poor recovery of packaged phage DNA as reflected in the low intensity of the parental band.

DNA isolated from the packaged sham controls

produces strong parental bands, but virtually no heteroduplex bands, indicating that unpackaged heteroduplex DNA was successfully removed by the phage purification steps. A faint 1.7-kb band, as well as an equally faint 1.0-kb band, can be seen in lanes overloaded with digests of packaged sham control DNA. These t w o bands would be expected with a low level

of incomplete Hind111 digestion of the sham DNA

itself. I t is unlikely that the very faint heteroduplex band observed in the case of the packaged 26 bp het DNA is a partial restriction fragment, however, since the digest was clearly not overloaded, and the 1.0-kb partial restriction fragment is absent.

Digestion of the total DNA isolated from the 19-bp and 26-bp het DNA packaging extracts at the end of the 2-hr incubation gives both 0.56-kb and 1.7-kb bands at approximately the same relative intensities. Thus, there does not appear to be a selective loss of heteroduplex material during these in vitro packaging incubations. In contrast, the heteroduplex band is less intense relative to the parental band in the case of the 690-bp het DNA packaging extracts. This effect was also seen with DNA isolated from another packaging extract in which a different preparation of 690-bp het DNA was used (data not shown). T h e 690-bp het DNA also displayed a frequency of heterozygous products of transfection lower than that expected on the basis of random assortment of strands. These observations suggest the possibility that heteroduplex chromosomes harboring a 690-bp heterology are subject to degradation.

DISCUSSION

Bacteriophage lambda chromosomes harboring cl

heterologies of up to 19 bp were packaged in vitro and found to produce heterozygous c l - / c I + phage

-Probe

cl289 or cl290

- M x A + + W ~ +

(+l9 bp or + 26 bp) + o ~ + o . 5 s ~ ~ - 1 . s * D - ! FIGURE 2.-Restriction Map of

Xcl Homoduplex and Heteroduplex

0. k - I . ? &

-

DNA. Both +19-bp and +26-bp in-

Msp I Hind 111 Mspl sertions have XhoI linkers in the cl

Mspl HndIII Xhol

+

*

+*

W T Hind111 site. Parental DNA from

XclAH3 is deleted for the probe se-

quences. A restriction fragment har- boring the large heteroduplex loop created by the 690-bp deletion mi- grates aberrantly in agarose gels as a Msp I Hnd 111 HIM 111 Msp I

c'AM

-

'1.9 kb'

-

(- 690 bp) 0 4 5 k -1,Okb- MW I Msp I

Msp I Htrd Ill Msp I 1.9-kb fragment.

Parental Chromosomes

Heteroduplex

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among the packaged products. Thus, DNA that in- cludes small heterologies can be packaged successfully. Phage packaged with DNA harboring a base pair mismatch or with DNA harboring heterologies of up to 19 bp yield similar frequencies of heterozygotes,

showing that phage DNA containing a heterology as

large as 19 bp is packaged nearly as efficiently as is phage DNA harboring only base pair mismatches. In contrast, the yield of heterozygotes is greatly reduced when a heterology of 26 bp is present. With a 690 bp heterology, no heterozygotes are observed above the background level of detection. T h e relative yield of packaged heterozygotes was assessed by measuring the frequency of heterozygosity at c l and heterozygosity at 0 and P in the packaged phage, as well as by

Southern analysis of packaged phage DNA. There

was a 10- to 20-fold reduction in the frequency of c l

heterozygotes and of 0-P heterozygotes packaged

with DNA harboring a 26-bp heterology as compared

to heterozygotes packaged with a 19-bp heterology. Southern analysis of packaged phage similarly shows evidence of a large reduction in the amount of heterology-containing DNA when the size of the heter-

ology increases from 19 to 26 bp. Thus, DNA har-

boring a 26-bp heterology appears to be encapsidated much less efficiently than DNA with a modestly smaller heterology.

What is the basis of the difference in packaging efficiency of DNA containing a 19-bp heterology as compared to DNA containing a 26-bp heterology? One possibility is that the size of the larger heterology physically interferes with the packaging process. It would appear that during encapsidation, the phage DNA passes through the head-tail connector into the

prohead (KOCHAN and MURIALDO 1983; TSUI and

HENDRIX 1980), raising the possibility that structural

perturbations in the DNA duplex could block passage through the portal of the connector. Translocation of DNA into the phage T 4 prohead, for example, appears to be blocked by the presence of branches in the phage DNA substrate, which must first be re-

moved by the action of T 4 endonuclease VI1 (MIZU-

UCHI et al. 1982; HSIAO and BLACK 1978; KEMPER

and JANZ 1976; KEMPER and BROWN 1976). T h e

dimensions of the portal would naturally limit the size of heterology that could be packaged, and this could account for the cut-off between packaging DNA with 19 bp and 26 bp heterologies. T h e diameter of the portal has been estimated to be similar to the width

of the DNA double helix (KOCHAN, CARRASCOSA and

MURIALDO 1984). It is therefore surprising that even

a 19-bp heterology can be accommodated in packaging. It may be that the portal is dilated during packaging, thus allowing the smaller heterologies to get through. In other variations of this model, large heterologies could interfere with the packaging apparatus

or a packaging monitoring mechanism (SYVANEN and

YIN 1978; FEISS and WIDNER 1982); for example, if

terminase and/or some monitoring protein traverses the DNA during packaging, the 19-bp heterology

might be tolerated while a 26-bp heterology might

not. A larger portal size in the case of phage T4 could

perhaps accommodate the evidence of Mosrc and

POWELL (1985), of packaging of DNA with a 150-bp

heterology when the gene 49 (endonuclease VII)

product is disabled.

Another possibility to account for the boundary between 19-bp and 26-bp heterologies could be the presence of a DNA-binding protein in the crude packaging extract that recognizes and binds only heterol-

ogies of 26 bp or more. This bound protein could

physically prevent entry of the DNA into the prohead. It is also possible that the 26-bp heterology could be selectively repaired or degraded, but this seems unlikely since Southern analysis of DNA isolated from the packaging extract at the end of the two hour incubation does not indicate a selective loss of the 26-

bp heterology-containing restriction fragment. How-

ever, one cannot rule out the possibility that the larger heterologies are enzymatically nicked on one strand, since a nicked DNA fragment would migrate to the same position as the intact fragment in a gel. Evidence obtained with phage T 4 suggests that nicked DNA

substrates are not packageable (ZACHARY and BLACK

1981). This class of models, in which a DNA-binding protein selectively binds and/or nicks heterologies of

26 bp or more, seems less attractive than models

postulating that the larger heterologies physically ob- struct DNA translocation.

T h e in vitro packaging of each het DNA preparation

yields a lower frequency of heterozygotes among pack-

aged products than expected on the basis of transfec-

tion. For example, cZ60 het DNA was prepared by

denaturing and reannealing DNA from XcZ+ Pam80

with DNA from XcI60 Oam29; it therefore contains

heteroduplex molecules which would harbor three

base mismatches (at cZ60, Pam80 and Oam29), as well

as parental homoduplex chromosomes. As indicated by the frequency of

CZ

heterozygous products of transfection, the cI60 het DNA preparation had approximately 42% heteroduplex molecules, as expected on the basis of random assortment of annealed strands. However, only 13% of the phage packaged with this DNA are heterozygous for cZ, suggesting that the mismatches may have interfered with the encapsidation of the heteroduplex molecules. Perhaps similarly, the presence of pyrimidine dimers in bacteriophage T 4 DNA appears to impair the in vivo encapsidation

of the phage DNA, although some dimers are ulti-

mately packaged (ZACHARY and BLACK 1984). It

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Packaging DNA Heterologies in h 1 1

sult of the disruption in the DNA duplex caused by

the mismatch, since a heterology of 19 bp, which

represents a much greater disruption, is packaged to nearly the same extent. It is also possible that heterologies, including mismatches, are targets of endonuclease activity during incubation in the extract. Such an activity might cause the selective loss of heteroduplex chromosomes. Another perhaps related possibility could be the presence of a protein in the extract that binds to DNA mismatches, thereby impairing

encapsidation of the heteroduplex DNA. In these

models, all heteroduplexes would be impaired in pack-

aging to about the same extent, but homoduplex DNA

molecules, i.e., parental chromosomes, would be un-

impaired.

Although in vitro packaging has been the focus of the experiments described here, sotne interesting genetic implications are evident. First, as seen in Table

5 , phage packaged with DNA harboring heterologies

of 19 bp or less all give high titers of O+P+ plaques on the su- mutL indicator strain. T h e packaged hetero-

zygotes have mismatched 0 and P markers and cannot

give rise to O+P+ plaques unless corrected by mis-

match repair. One might have expected that the mutL

indicator, which is deficient in mismatch correction

(Lu, CLARK and MODRICH 1983; WAGNER et al. 1984),

would not give a high frequency of O+P+ plaques

from

0-P

heterozygotes. HUISMAN and Fox (1986),

for example, observed only about 10% mismatch-

correction of heterozygotes in a mutL host that was not selective. However, in the case described here, the

0-P

heterozygotes cannot replicate, and this could

allow time for more extensive correction by the resid- ual mismatch repair activity in the mutL indicator. It was also observed that the O+P+ titers of packaged heterozygous phage were 1 0-fold lower on an su-mut+

indicator than on the su- mutL indicator. Although the level of mismatch correction would be expected

to be higher in the wild-type indicator, it is also likely that the repair tracts could be considerably longer in this host (FOX and RAPOSA 1983). Co-correction of

both 0 and P mismatches would reduce the yield of

O+P+ plaques.

I t is also surprising that packaged heterozygous phage display such high yields of recombinants on the

su- mutL indicator. For example, it would appear that

14% of the phage packaged with the 19-bp het DNA

yield O+P+ plaques. Transfection and the results of

Southern analysis indicate that about 10% of these

same packaged phage are heterozygous for GI. Thus,

it appears as though all of the heterozygous phage

would have to produce O+P’ plaques on the su- mutL

indicator. In other words, we are led to the unlikely possibility that all mismatch repair events restore the wildtype alleles on the transcribed strand. It is possible that the high frequency of O+P+ plaques is only ap-

parent for the following reason. T h e “frequency” of

O+P+ plaques among packaged phage reflects the plating efficiencies of the packaged phage on two different hosts, su- and sull. If the efficiency of plating

of the packaged phage were for some reason low on

the sull strain, then the ratio of the titers on selective and permissive indicators would not reflect the rela-

tive proportion of heterozygous phage among the

packaged products. It is possible, for example, that as the result of the action of nucleases in the in vitro

packaging extracts or the process of denaturation and

reannealing, DNA damages are present in the packaged phage. Suppression of the amber mutations in the permissive host could result in replication of phage DNA before the damages had been repaired, and result in loss of viability of the products. In the selective host, however, no replication could occur until the 0 or P mismatch was corrected to wild type. T h e resulting delay in replication might permit repair of DNA damages, so that more phage DNA survives as an intact product. Thus, the sull titer may be a dis- torted reflection of the total yield of infectious packaged phage.

We have shown that small heterologies can be packaged in vitro to give viable heterozygous progeny and that the size of the heterology that can be packaged is subject to a boundary condition. T h e results of the

recombination experiments presented previously

(PEARSON and FOX 1988) corroborate these in vitro

packaging results, and suggest a discontinuity in packaging competence both in vivo and in vitro when the size of a heterology increases from 19 to 26 bp.

We thank BOB BOURRET and DAVE PEARSON for their help in preparing this and the accompanying manuscript. R.K.P. was sup- ported by training grant GM07287 from the National Institutes of Health and by a duPont Graduate Fellowship. This work was supported by National Institutes of Health grant A105388 to M.F.

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