Nat Sternberg

In vitro packaging of a λ Dam vector containing EcoRI DNA fragments of Escherichia coli and phage P1

Abstract In this report we describe a coliphage λ vector system for cloning endo R. Eco RI DNA fragments. This system differs significantly from those previously described in two ways. First, restricted and ligated DNA is encapsidated in vitro. Second, with increasing λ DNA size in the range 78 to 100% that of wild-type, the efficiency of DNA encapsidation into infectious phage particles markedly increases. For λ wild-type DNA the efficiency of in vitro packaging (10 6 to 10 7 plaques produced per μg of added DNA) is equal to, or better than, the standard CaCl 2 transfection method. The use of a D am mutation to facilitate recognition of size classes of inserted fragments is described. Using this vector and in vitro packaging, several E. coli and phage P1 endo R. Eco RI fragments were cloned.

Packaging of coliphage lambda DNA. II. The role of the gene D protein.

Abstract The gene D protein (p D ) of coliphage λ is normally an essential component of the virus capsid. It acts during packaging of concatemeric λ DNA into the phage prohead and is necessary for cutting the concatemers at the cohesive end site ( cos ). In this report we show that cos cutting and phage production occur without p D in λ deletion mutants whose DNA content is less than 82% that of λ wild type. D -independence appears to result directly from DNA loss rather than from inactivation (or activation) of a phage gene. (1) In cells mixedly infected with undeleted λ and a deletion mutant, particles of the deletion mutant alone are efficiently produced in the absence of p D ; and (2) D -independence cannot be attributed to loss of a specific segment of the phage genome. p D -deficient phage resemble p D -containing phage in head size and DNA ends; they differ in their extreme sensitivity to EDTA, greater density, and ability to accept p D . p D appears to act by stabilizing the head against disruption by overfilling with DNA rather than by changing the capacity of the head for DNA. This is shown by the observation that the amount of DNA packaged by a “headful” mechanism, normally in excess of the wild-type chromosome size, is not reduced in the absence of p D . In fact, p D is required for packaging headfuls of DNA. This implies that a mechanism exists for preventing the entry of excess DNA into the head during packaging of concatemers formed by deletion mutants, and we suggest that this is accomplished by binding of cos sites to the head. The above results show that p D is not an essential component of the nuclease that cuts λ concatemers at cos during packaging, and they imply that 82% of a wild-type chromosome length can enter the prohead in the absence of p D . Yet, p D is needed for the formation of cohesive ends after infection with undeleted phage. We propose two models to account for these observations. In the first, cos cutting is assumed to occur early during packaging. The absence of p D leads to release of packaged DNA and the loss of cohesive ends by end-joining. In the second, cos cutting is assumed to occur as a terminal event in packaging. p D promotes cos cutting indirectly through its effect on head stability. We favor the second model because it better explains the asymmetry observed in the packaging of the chromosomes of cos duplication mutants (Emmons, 1974).

A genetic analysis of bacteriophage λ head assembly

Abstract The sequence of steps involved in bacteriophage λ head assembly has been studied by characterizing second-site mutatons that can compensate for the reduced level of synthesis of a particular phage “head” protein. Such studies reveal these classes of protein interactions: (1) A reduced level of either p A synthesis or activity can compensate for the reduced amount of p D made when λD am mutants are grown in a supC host. This result implies that p A acts before p D during the packaging of phage DNA. (2) A reduced level of either p B synthesis or activity can compensate for the reduced amount of p E made when λE am mutants are grown in a sup C host, and vice versa. This result suggests an interaction between these proteins that is stringently dependent on maintaining a proper ratio between them. An identical argument has been made to explain the properties of gro E hosts (Sternberg, 1973) . (3) A very specific class of mutations in phage gene E can compensate for the reduced amount of p C made when λC am mutants are grown in a sup C host. This suggests that the products of these two genes directly interact. Using the techniques described in this report it is possible to easily isolate a variety of mutations ( am, oc, ts, and missense) in specific phage “head” genes (genes A, B and E).

Packaging of coliphage lambda DNA: I. The role of the cohesive end site and the gene A protein

Abstract The cohesive ends of the DNA of bacteriophage λ particles are normally formed by the action of a nuclease on the cohesive end sites (cos) of concatemeric λ DNA (reviewed by Hohn et al., 1977). The nuclease also cuts the cos site of an integrated prophage, and DNA located to the right is preferentially packaged into phage particles. This process occurs with approximately the same efficiency and rate in a single lysogen as in a tandem polylysogen. Thus, the rate of cos cutting does not increase when the number of cos sites per molecule increases, an hypothesis that has been proposed to explain why cohesive ends are not formed in circular monomers of λ DNA. We propose instead that the interaction of Ter with cos is influenced by the configuration of the DNA outside of cos during packaging, and that this configuration is different for circular monomers than for other forms of λ DNA. A model that gives rise to such a difference is described. We also found that missense mutations in the λ A gene changed the efficiency of packaging of phage relative to host DNA. This was not the case for missense mutations in several phage genes required for capsid formation. Thus, the product of gene A plays a role in determining packaging specificity, as expected if it is or is part of the nuclease that cuts λ DNA at cos.

A characterization of bacteriophage P1 DNA fragments cloned in a λ Vector

Abstract Seventeen of the twenty-six possible EcoRI-generated fragments of P1 DNA have been cloned in a λD−srIλ3 vector. The results of marker rescue experiments with these λ-P1 hybrid phages reveal a clustering of P1 am mutations on the P1 DNA and a localization to a small part of that DNA of P1 functions involved in plasmid replication and plasmid segregation fidelity. Various elements of the complex P1 immunity system have been mapped to particular P1 fragments located in three separate regions of the P1 genome. Hybrid phages that express the P1 ban gene and P1 modification (mod) gene have also been isolated. The recombinant phages described in this paper constitute a set of tools with which to define the role of particular segments of the P1 genome in the physiology of this organism.

A class of rifR RNA polymerase mutations that interferes with the expression of coliphage λ late genes

Abstract This report describes the isolation and characterization of a class of RNA polymerase ( rif R ) mutations in Escherichia coli strain C600 that retards the growth of bacteriophage λ. In a typical example of this class of mutants, strain C600 rif R -2, phage early functions ( p N, pint, pxis , phage DNA replication, and phage early mRNA synthesis) are expressed normally. However, the expression of late phage functions (late mRNA synthesis, lysozyme synthesis, and virus production) is delayed in this host. Since the transcription of late λ genes depends partially on the prior synthesis of phage gene Q protein ( p Q), this defect could reflect either an inability of p Q to work properly in the mutant host (activity model) or reduced p Q synthesis in this host (synthesis model). The synthesis model is supported by two results: (1) phage growth in the absence of p Q is also defective in the mutant host; and (2) phage mutations, nin 5 and byp , which bypass the need for p N at transcription termination signal t R2 overcome the host defect. Thus, this defect must at least in part reflect a reduced ability of p N to allow transcription beyond termination signal t R2 into the Q gene. However, it cannot be ruled out that the rif R -2 mutation might also affect p Q activity. Since p N works normally in the mutant host to overcome transcription barriers at sites t R1 and t L , the host RNA polymerase mutation appears to affect the ability of p N to act at the various phage transcription termination signals differentially. A strain containing both the rif R -2 mutation and a second host mutation defective for phage p N activity, nus A-1 (Friedman et al. , 1973a), has been constructed and is significantly more defective for phage growth than can be accounted for by the additive defects produced by both single mutations. This synergistic effect suggests that proper interaction between at least two host components (RNA polymerase and pnus A) and a phage component ( p N) is essential for normal transcription of the λ genome.

Altered arrangement of the DNA in injection-defective lambda bacteriophage

Abstract We have characterized two variant bacteriophage λ particles, λZ− and λdoc L, that have low infectivity but normal morphology. The low infectivity is due, at least in part, to a defect in DNA injection. This defect is probably the result of an altered location of the right end of the chromosome with respect to the phage tail: the right end of λZ− and λdoc L DNA, in contrast to that of wild-type λ, cannot be cross-linked to the tail. The cross-linking experiments were greatly facilitated by a new technique that allows routine spreading of DNA for electron microscopy without the use of a protein film. We propose that the Z gene product, a tail protein, acts by recognizing a specific feature near the right terminus of the DNA and promoting its insertion into the tail. This feature is presumably missing in most λdoc L particles.

Transduction of recB- Hosts is Promoted by λ red + Function

The redB and redX genes of bacteriophage λ specify a recombination pathway that efficiently recombines phage genomes in lytically infected recA- and recB- cells (see Signer, 1971, for a review). However, the red genes do not promote efficient specialized transduction of a recA- host (Manly et al., 1969; Mizuuchi and Fukasawa, 1969; Gottesman et al., 1974; H. Echols, personal communication). We report here that the red pathway does promote efficient transduction of redB- hosts.