Phage T1-mediated transduction of a plasmid containing the T1 pac site

The T1 pac site has been cloned into a plasmid vector. This recombinant plasmid was tested for T1-mediated transduction efficiency in comparison with a plasmid containing the phage lambda T1-pac-like site esp-lambda, plasmids containing T1 sequences other than the pac site, and plasmids containing neither T1 sequences nor known pac sites. The data obtained indicate that there are at least two distinct mechanisms of T1-mediated plasmid transduction. One requires the presence of any T1 sequence on the plasmid and probably takes place via cointegrate formation with the homologous region of an infecting T1 genome. The other is specifically dependent on the presence of a pac site on the plasmid. Plasmids are packaged as head-to-tail multimers that have one heterogeneous molecular end and the other terminated at pac, and the direction of packaging with respect to the pac site is the same for plasmids as for T1. Possible roles of pac in plasmid packaging and their implications with regard to the packaging of phage DNA are discussed.     

The last duplex base-pair of the phage lambda chromosome. Involvement in packaging, ejection and routing of lambda DNA.

cosN is the site at which the bacteriophage lambda DNA packaging enzyme, terminase, introduces staggered nicks to generate the cohesive ends of mature lambda chromosomes. Genetic and molecular studies show that cosN is recognized specifically by terminase and that effects of cosN mutations on lambda DNA packaging and cosN cleavage are well correlated. Mutations affecting a particular base-pair of cosN are unusual in being lethal in spite of causing only a moderate defect in cosN cleavage and DNA packaging. The particular base-pair is the rightmost duplex base-pair in mature chromosomes, at position 48,502 in the numbering system of Daniels et al; herein called position - 1. A G.C to T.A transversion mutation at position - 1, called cosN - 1T, reduces the particle yield of lambda fivefold, and the particles formed are not infectious. lambda cosN - 1T particles have wild-type morphology, and contain chromosomes that have normal cohesive ends. The chromosomes of lambda cosN - 1T particles, like the chromosomes of lambda + particles, are associated with the tail. lambda cosN - 1T particles, in spite of being normal structurally, are defective in injection of DNA into a host cell. Only approximately 25% of lambda cosN - 1T particles are able to eject DNA from the capsid in contrast to 100% for lambda +. Furthermore, for the 25% that do eject, there is a further injection defect because the ejected lambda cosN - 1T chromosomes fail to cyclize, in contrast to the efficient cyclization found for wild-type chromosomes following injection. The cosN - 1T mutation has no effect on Ca2+ mediated transformation by lambda DNA, indicating that the effect of the mutation on DNA fate is specific to the process of DNA injection. Models in which specific DNA : protein interactions necessary for DNA injection, and involving the rightmost base-pair of the lambda chromosome, are considered.     

Sites and gene products involved in lambdoid phage DNA packaging.

21 is a temperate lambdoid coliphage, and the genes that encode the head proteins of lambda and 21 are descended from a common ancestral bacteriophage. The sequencing of terminase genes 1 and 2 of 21 was completed, along with that of a segment at the right end of 21 DNA that includes the R4 sequence. The R4 sequence, a site that is likely involved in termination of DNA packaging, was found to be very similar to the R4 sequences of lambda and phi 80, suggesting that R4 is a recognition site that is not phage specific. DNA packaging by 21 is dependent on a host protein, integration host factor. A series of mutations in gene 1 (her mutations), which allow integration host factor-independent DNA packaging by 21, were found to be missense changes that affect predicted alpha-helixes in gp1. gp2, the large terminase subunit, is predicted to contain an ATP-binding domain and, perhaps, a second domain important for the cos-cutting activity of terminase. orf1, an open reading frame analogous in position to FI, a lambda gene involved in DNA packaging, shares some sequence identity with FI. orf1 was inactivated with nonsense and insertion mutations; these mutations were found not to affect phage growth. 21 was also not able to complement a lambda FI mutant.     

Transduction of bacteriophage lambda by bacteriophage T1.

When bacteriophage T1 was grown on bacteriophage lambda-lysogenic cells, phenotypically mixed particles were formed which had the serum sensitivity, host range, and density of T1 but which gave rise to lambda phage. T1 packaged lambda genomes more efficiently both when the length of the prophage was less than that of wild-type lambda and when the host cell was polylysogenic. Expression of the red genes of lambda or the recE system of Escherichia coli during T1 growth enhanced pickup of lambda by T1, whereas packaging was reduced in recB cells. If donors were singly lysogenic, the expression of transduced lambda genomes as a PFU required lambda-specified excisive recombination, whereas lambda genomes transduced from polylysogens required only lambda- or E. coli-specified general recombination to give a productive infection.     

The interaction of Escherichia coli integration host factor with the cohesive end sites of phages lambda and 21

The interaction of E. coli integration host factor (IHF) with the cohesive end sites (cos's) of phages lambda and 21 has been studied by the DNAase I footprinting technique. Six potential sites in cos lambda differ from the consensus IHF binding sequence by 1 to 3 base pairs. Of the six, one site, I1, binds IHF strongly. The I1 segment protected by IHF contains two sequences that closely match the IHF consensus binding sequence. Another site, I2, binds IHF moderately well, and three sites: 10', 13 and 14 bind IHF very weakly. The 10 site does not bind IHF under the conditions used here. In phage 21 the DNA segment extending to the right from the cohesive ends, which contains three potential IHF binding sites, was examined. Two sites bind IHF well; I1, the 21 analogue of one of the lambda I1 sites, and I0, a site not analogous to a lambda site. The third 21 site, I2, binds IHF moderately well, as does the analogous I2 site in lambda. The significance of the results for lambda DNA packaging is discussed.     

Identification of sequences necessary for packaging DNA into lambda phage heads

Several species of DNA molecules are packaged into lambda phage heads if they carry the region around the cohesive end site of lambda phage (cos lambda). The minimal functional sequence around cos lambda needed for packaging was examined by cloning in pBR322. The results showed that the minimal region contained 85 bp around cos lambda; 45 bp of the left arm of lambda phage and 40 bp of the right arm. A 75-bp region located to the right of the minimal region seems to enhance packaging. A 223-bp fragment containing these regions can be used as a portable element for plasmid DNA packaging into lambda phage heads. Plasmid ppBest 322, a derivative of pBR322 carrying this portable packager and both amp and tet genes, was constructed. This plasmid is useful for cloning of large DNA fragments.     

The Nu1 subunit of bacteriophage lambda terminase

The maturation and packaging of bacteriophage lambda DNA are catalyzed by the phage terminase enzyme. Terminase is composed of two protein subunits, gpNu1 and gpA. The holoenzyme is multifunctional in vitro; it binds to and cleaves lambda DNA at the cos site (where cos represents cohesive-end site), packages DNA into lambda proheads, and is also a DNA-dependent ATPase. The genes of the two subunits have been cloned separately into powerful expression vectors which allow for very high levels of protein overproduction. The gpNu1 protein has been purified to homogeneity and has a monomeric molecular weight of 21,200, in close agreement with the Mr of 20,444 expected from its amino acid sequence. Both gel filtration and sedimentation velocity centrifugation indicate that the native gpNu1 protein exists as a Mr greater than 500,000 aggregate. The sequence of the first 20 amino acids and the overall composition both match those predicted by the nucleotide sequence of the Nu1 gene. Purified gpNu1 is able to complement gpA-containing extracts in both lambda DNA packaging and cos cleavage assays. The Nu1 gene amino acid sequence predicts DNA binding by the protein, and gpNu1 does show specific binding to lambda DNA by filter binding assays. Also, as predicted from its sequence, gpNu1 exhibits ATPase activity; but in contrast to the holoenzyme, this activity is DNA-independent.     

Bacteriophage lambda gpNu1 and Escherichia coli IHF proteins cooperatively bind and bend viral DNA: implications for the assembly of a genome-packaging motor

Terminase enzymes are common to both prokaryotic and eukaryotic double-stranded DNA viruses and are responsible for packaging viral DNA into the confines of an empty procapsid shell. In all known cases, the holoenzymes are heteroligomers composed of a large subunit that possesses the catalytic activities required for genome packaging and a small subunit that is responsible for specific recognition of viral DNA. In bacteriophage lambda, the DNA recognition protein is gpNu1. The gpNu1 subunit interacts with multiple recognition elements within cos, the packaging initiation site in viral DNA, to site-specifically assemble the packaging machinery. Motor assembly is modulated by the Escherichia coli integration host factor protein (IHF), which binds to a consensus sequence also located within cos. On the basis of a variety of biochemical data and the recently solved NMR structure of the DNA binding domain of gpNu1, we proposed a novel DNA binding mode that predicts significant bending of duplex DNA by gpNu1 (de Beer et al. (2002) Mol. Cell 9, 981-991). We further proposed that gpNu1 and IHF cooperatively bind and bend viral DNA to regulate the assembly of the packaging motor. Here, we characterize cooperative gpNu1 and IHF binding to the cos site in lambda DNA using a quantitative electrophoretic mobility shift (EMS) assay. These studies provide direct experimental support for the long presumed cooperative assembly of gpNu1 and IHF at the cos sequence of lambda DNA. Further, circular permutation experiments demonstrate that the viral and host proteins each introduce a strong bend in cos-containing DNA, but not nonspecific DNA substrates. Thus, specific recognition of viral DNA by the packaging apparatus is mediated by both DNA sequence information and by structural alteration of the duplex. The relevance of these results with respect to the assembly of a viral DNA-packaging motor is discussed.     

The bacteriophage lambda terminase. Partial purification and preliminary characterization of properties

The maturation of bacteriophage lambda DNA and its packaging into preformed heads to produce infectious phage is under the control of the two leftmost genes on the lambda chromosome, i.e., Nu1 and A. Based on its ability to complement lambda A- phage-infected cell extracts for packaging of lambda DNA in vitro, a single protein, designated terminase (ter) has been extensively purified using adsorption, ion exchange, and affinity column chromatography. The final preparation represents an approximately 60,000-fold purification over the activity found in crude extracts and is about 30 to 80% homogeneous as judged by visualizing the protein after electrophoresis in sodium dodecyl sulfate-polyacrylamide gel. In addition to packaging, terminase can also catalyze the endonucleolytic cleavage of lambda cohesive-end site DNA; both of these reactions require ATP. In some preparations, certain terminase fractions of extreme purity require protein factors present in extracts of uninfected Escherichia coli in order to catalyze the cohesive-end site cleavage reaction. On ion exchange columns purified terminase co-chromatographs with a DNA-dependent ATPase activity, hydrolyzing ATP to ADP and Pi in the presence of any of several types of DNA tested including those of non-lambda origin. The molecular weight of the native enzyme is 117,000 and appears to be a hetero-oligomer composed of 2 nonidentical subunits. The most likely composition of terminase is one gpA (gene product of A), Mr = 74,000 and two gpNu1, Mr = 21,000.     

An accessory role for Escherichia coli integration host factor: characterization of a lambda mutant dependent upon integration host factor for DNA packaging.

Bacteriophage lambda grows lytically on Escherichia coli defective for integration host factor, a protein involved in lambda site-specific recombination and the regulation of gene expression. We report the characterization of a mutant, lambda cos154, that, unlike wild-type lambda, is defective for growth in integration host factor-defective E. coli. The cis-dominant mutation in lambda cos154 is a single base pair change in a region of hyphenated dyad symmetry close to the lambda left cohesive end; this mutation prevents DNA packaging. We propose the following two alternative roles for this site in lambda DNA packaging: (i) to bind an E. coli accessory protein required in the absence of integration host factor or (ii) to bind the phage-encoded terminase protein that is essential for DNA packaging.     

Nucleotides regulate the conformational state of the small terminase subunit from bacteriophage lambda: implications for the assembly of a viral genome-packaging motor

Terminase enzymes are responsible for "packaging" of viral DNA into a preformed procapsid. Bacteriophage lambda terminase is composed of two subunits, gpA and gpNu1, in a gpA(1).gpNu1(2) holoenzyme complex. The larger gpA subunit is responsible for preparation of viral DNA for packaging, and is central to the packaging motor complex. The smaller gpNu1 subunit is required for site-specific assembly of the packaging motor on viral DNA. Terminase assembly at the packaging initiation site is regulated by ATP binding and hydrolysis at the gpNu1 subunit. Characterization of the catalytic and structural interactions between the DNA and nucleotide binding sites of gpNu1 is thus central to our understanding of the packaging motor at the molecular level. The high-resolution structure of the DNA binding domain of gpNu1 (gpNu1-DBD) was recently determined in our lab [de Beer, T., et al. (2002) Mol. Cell 9, 981-991]. The structure reveals the presence of a winged-helix-turn-helix DNA binding motif, but the location of the ATPase catalytic site in gpNu1 remains unknown. In this work, nucleotide binding to the gpNu1-DBD was probed using acrylamide fluorescence quenching and fluorescence-monitored ligand binding studies. The data indicate that the minimal DBD dimer binds both ATP and ADP at two equivalent but highly cooperative binding sites. The data further suggest that ATP and ADP induce distinct conformations of the dimer but do not affect DNA binding affinity. The implications of these results with respect to the assembly and function of a terminase DNA-packaging motor are discussed.     

Genetic analysis of cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda.

cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda, consists of three binding sites (called R3, R2 and R1) for gpNu1, the small subunit of terminase; and I1, a binding site for integration host factor (IHF), the DNA bending protein of Escherichia coli. cosB is located between cosN, the site where terminase introduces staggered nicks to generate cohesive ends, and the Nu1 gene; the order of sites is: cosN-R3-I1-R2-R1-Nu1. A series of lambda mutants have been constructed that have single base-pair C-to-T transition mutations in R3, R2 and R1. A single base-pair transition mutation within any one of the gpNul binding sites renders lambda dependent upon IHF for plaque formation. lambda phage with mutations in both R2 and R3 are incapable of plaque formation even in the presence of IHF. Phages that carry DNA insertions between R1 and R2, from 7 to 20 base-pairs long, are also IHF-dependent, demonstrating the requirement for a precise spacing of gpNu1 binding sites within cosB. The IHF-dependent phenotype of a lambda mutant carrying a deletion of the R1 sequence indicates that IHF obviates the need for terminase binding to the R1 site. In contrast, a lambda mutant deleted for R2 and R1 fails to form plaques on either IHF+ or IHF- cells, indicating terminase binding of R2 is involved in suppression of R mutants by IHF. A fourth R sequence, R4, is situated on the left side of cosN; a phage with a mutant R4 sequence shows a reduced burst size on both an IHF+ and an IHF- host. The inability of the R4- mutant to be suppressed by IHF, plus the fact that R4 does not bind gpNu1, suggests R4 is not part of cosB and may play a role in DNA packaging that is distinct from that of cosB.     

Packaging of ColE1 DNA having a lambda phage cohesive end site.

The mechanism of lambda phage-mediated transduction of hybrid colicin E1 DNAs of various lengths was studied, and factors influencing the formation of these transducing particles were investigated. The results were as follows: 1. The presence of a cohesive end site of lambda phage (coslambda) on colicin E1 DNA was essential for packaging of the DNA. 2. Packaging of colicin E1 DNAs, which carry coslambda with molecular sizes corresponding to 68% of that of lambda phage DNA, was observed in the absence of all known recombination functions of E. coli K-12 and of lambda phage. 3. Hybrid colicin E1 DNAs having coslambda with molecular sizes corresponding to 28% of that of lambda phage DNA were packaged within lambda phage particles as trimers; hybrid DNAs with coslambda of 40 and 47% of the length of lambda phage DNA were packaged as dimers; and those with molecular sizes of 68% of that of lambda phage DNA were packaged mostly as monomers. These results demonstrated that two factors are essential for the packaging of DNAs within lambda phage particles; the presence of coslambda on the DNA molecule and an appropriate size of DNA.     

Structure of the bacteriophage lambda cohesive end site: location of the sites of terminase binding (cosB) and nicking (cosN)

The extents of the sites for nicking (cosN) and binding (cosB) of bacteriophage lambda DNA by terminase have been determined by studying cos cleavage and terminase binding in vitro. The cosN site is located in the segment from -22 to +24 bp (numbered from the center of the cohesive end sequence in the circular lambda genome). The cosB site is located in the segment from +51 to +120 (the +120 boundary determined by Miwa and Matsubara, 1983). Additional sequences are necessary for packaging into infectious phage particles, including regions to the left (Rz gene side) of cosN, and between cosN and cosB. Small deletions (7 and 11 bp) between cosN and cosB abolish packaging in vivo without affecting cos binding and cleavage in vitro, whereas a large deletion (26 bp) abolishes packaging in vivo and cleavage in vitro.     

A Novel Method for Generating Nested Deletions Using the in Vitro Bacteriophage T3 DNA Packaging System

To sequence a DNA segment inserted into a cosmid vector underthe directed sequencing strategy, we established a simple andrapid method for generating nested deletions which uses thein vitro packaging system of bacteriophage T3 DNA. The principleis based on the previous finding that this system can translocateany linear double-stranded DNA up to 40 kb into the phage capsidin a time-dependent manner and the encapsulated DNA becomesDNase-resistant. For this purpose, we constructed a cosmid vectorthat carries two different antibiotic selection markers at bothsides of the multiple cloning site, and after insertion of aDNA segment, the clone was linearized by -terminase at the cossite. After the packaging reaction in vitro followed by DNasetreatment, the encapsulated DNA was introduced into Escherichiacoli cells to give clones with unidirectional deletions by differentialantibiotic selection. Restriction and sequence analyses of deletionclones demonstrated that an ordered set of clones with nesteddeletions, ranging from less than 1 kb to 25 kb, was createdfrom either the end of the DNA segment. Thus, nested deletionclones that cover the entire region of a 40-kb cosmid insertcan be obtained by a single packaging reaction, and its restrictionmap can be simultaneously obtained.     

Gene 19 of bacteriophage T7. Overexpression, purification, and characterization of its product

Gene 18 and 19 proteins of bacteriophage T7 are essential for DNA maturation and packaging. The phage capsid is the site of both maturation and packaging of T7 DNA. Both gene 18 and 19 proteins bind to capsid intermediates during DNA packaging but are not found in mature virions, suggesting that they play a direct role in the enzymatic mechanisms of DNA maturation and packaging. As part of an effort to reconstitute T7 DNA maturation and packaging with purified components, we have cloned and overexpressed T7 gene 19 in Escherichia coli. Gene 19 has been inserted downstream from the bacteriophage PL promoter controlled by the temperature-sensitive lambda repressor encoded by c1857. Upon thermal induction, most of the overproduced gene 19 protein is insoluble and inactive. However, by attenuation of the expression of gene 19 from the PL promoter, significant levels of soluble and active gene 19 protein are produced. Soluble gene 19 protein can be monitored by its ability to complement extracts of T7-infected cells for packaging of exogenous DNA. We have used this assay to monitor the activity of gene 19 protein during purification. The native protein is a monomer of molecular weight 66,000. We have also tested for the formation of a stable complex between gene 18 and 19 proteins. Coproduction of gene 18 and 19 proteins has no effect on either the solubility or activity of gene 19 protein, despite the fact that gene 18 protein is produced at at least 10-fold greater rates. Furthermore, we find no evidence for any interaction between soluble gene 18 and 19 proteins in extracts or between the purified proteins.     

Bacteriophage lambda DNA packaging: DNA site requirements for termination and processivity

Bacteriophage lambda chromosomes are processively packaged into preformed shells, using end-to-end multimers of intracellular viral DNA as the packaging substate. A 200 bp long DNA segment, cos, contains all the sequences needed for DNA packaging. The work reported here shows that efficient DNA packaging termination requires cos's I2 segment, in addition to the required termination subsite, cosQ, and the nicking site, cosN. Efficient processivity requires cosB, in addition to cosQ and cosN. An initiation-defective mutant form of cosB sponsored efficient processivity, indicating that the terminase-cosB interactions required for termination are less stringent than those required at initiation. The finding that an initiation-defective form of cosB is functional for processivity allows a re-interpretation of a similar finding, obtained previously, that the initiation-defective cosB of phage 21 is functional for processivity by the lambda packaging machinery. The cosBphi21 result can now be interpreted as indicating that interactions between cosBphi21 and lambda terminase, while insufficient for initiation, function for processivity.     

Defining the bacteriophage T4 DNA packaging machine: evidence for a C-terminal DNA cleavage domain in the large terminase/packaging protein gp17

Double-stranded DNA packaging in bacteriophage T4 and other viruses occurs by translocation of DNA into an empty prohead by a packaging machine assembled at the portal vertex. Coordinated with this complex process is the cutting of concatemeric DNA to initiate and terminate DNA packaging and encapsidate one genome-length viral DNA. The catalytic site responsible for cutting, and the mechanisms by which cutting is precisely coordinated with DNA translocation remained as interesting open questions. Phage T4, unlike the phages with defined ends (e.g. lambda, T3, T7), packages DNA in a strictly headful manner, and exhibits no strict sequence specificity to initiate or terminate DNA packaging. Previous evidence suggests that the large terminase protein gp17, a key component of the T4 packaging machine, possesses a non-specific DNA cutting activity. A histidine-rich metal-binding motif, H382-X(2)-H385-X(16)-C402-X(8)-H411-X(2)-H414-X(15)-H430-X(5)-H436, in the C-terminal half of gp17 is thought to be involved in the terminase cleavage. Here, exhaustive site-directed mutagenesis revealed that none of the cysteine and histidine residues other than the H436 residue is critical for function. On the other hand, a cluster of conserved residues within this region, D401, E404, G405, and D409, are found to be critical for function. Biochemical analyses showed that the D401 mutants exhibited a novel phenotype, showing a loss of in vivo DNA cutting activity but not the DNA packaging activity. The functional nature of the critical residues and their disposition in the conserved loop region between two predicted beta-strands suggest that these residues are part of a metal-coordinated catalytic site that cleaves the phosphodiester bond of DNA substrate. The data suggest that the T4 terminase consists of at least two functional domains, an N-terminal DNA-translocating ATPase domain and a C-terminal DNA-cutting domain. Although the DNA recognition mechanisms may be distinct, it appears that T4 and other phage terminases employ a common catalytic paradigm for phosphodiester bond cleavage that is used by numerous nucleases.     

Self-association properties of the bacteriophage lambda terminase holoenzyme: implications for the DNA packaging motor

Terminases are enzymes common to complex double-stranded DNA viruses and are required for packaging of viral DNA into a protective capsid. Bacteriophage lambda terminase holoenzyme is a hetero-oligomer composed of the A and Nu1 lambda gene products; however, the self-association properties of the holoenzyme have not been investigated systematically. Here, we report the results of sedimentation velocity, sedimentation equilibrium, and gel-filtration experiments studying the self-association properties of the holoenzyme. We find that purified, recombinant lambda terminase forms a homogeneous, heterotrimeric structure, consisting of one gpA molecule associated with two gpNu1 molecules (114.2 kDa). We further show that lambda terminase adopts a heterogeneous mixture of higher-order structures, with an average molecular mass of 528(+/-34) kDa. Both the heterotrimer and the higher-order species possess site-specific cos cleavage activity, as well as DNA packaging activity; however, the heterotrimer is dependent upon Escherichia coli integration host factor (IHF) for these activities. Furthermore, the ATPase activity of the higher-order species is approximately 1000-fold greater than that of the heterotrimer. These data suggest that IHF bending of the duplex at the cos site in viral DNA promotes the assembly of the heterotrimer into a biologically active, higher-order packaging motor. We propose that a single, higher-order hetero-oligomer of gpA and gpNu1 functions throughout lambda development.     

A functional domain of bacteriophage lambda terminase for prohead binding

Terminase is a multifunctional protein complex involved in DNA packaging during bacteriophage lambda assembly. Terminase is made of gpNul and gpA, the products of the phage lambda Nu1 and A genes. Early during DNA packaging terminase binds to lambda DNA to form a complex called complex I. Terminase is required for the binding of proheads by complex I to form a DNA: terminase: prohead complex known as complex II. Terminase remains associated with the DNA during encapsidation. The other known role for terminase in packaging is the production of staggered nicks in the DNA thereby generating the cohesive ends. Lambdoid phage 21 has cohesive ends identical to those of lambda. The head genes of lambda and 21 show partial sequence homology and are analogous in structure, function and position. The terminases of lambda and 21 are not interchangeable. At least two actions of terminase are involved in this specificity: (1) DNA binding; (2) prohead binding. The 1 and 2 genes at the left end of the 21 chromosome were identified as coding for the 21 terminase. gp1 and gp2 are analogous to gpNu1 and gpA, respectively. We have isolated a phage, lambda-21 hybrid 33, which is the product of a crossover between lambda and 21 within the terminase genes. Lambda-21 hybrid 33 DNA and terminase have phage 21 packaging specificity, as determined by complementation and helper packaging studies. The terminase of lambda-21 hybrid 33 requires lambda proheads for packaging. We have determined the position at which the crossover between lambda DNA and 21 DNA occurred to produce the hybrid phage. Lambda-21 hybrid 33 carries the phage 21 1 gene and a hybrid phage 2/A gene. Sequencing of lambda-21 hybrid 33 DNA shows that it encodes a protein that is homologous at the carboxy terminus with the 38 amino acids of the carboxy terminus of lambda gpA; the remainder of the protein is homologous to gp2. The results of these studies define a specificity domain for prohead binding at the carboxy terminus of gpA.