Jon Weil

Deletion analysis of two nonessential regions of the T4 genome

Abstract Mutants of T4 which contain a duplication of the rII region also often contain a compensating deletion of a nonessential segment of the genome. This relationship was used to isolate a series of viable deletion mutations. The deletions were mapped genetically, making use of their effect on plaque morphology, and by electron microscopy of DNA heteroduplexes to T2, a T2 T4 hybrid, and T4 wild type. They were also tested in several laboratories for various phage functions, enzymes, and proteins. Two different overlapping deletions located between genes 32 and 63 and ten located between genes 39 and 56 were obtained. Analysis of the former class indicates that there are at least 1400 base pairs of DNA between genes 32 and 63 which are unaccounted for by presently known genes. Deletions of the latter class affect the production of exonuclease A, a DNA-dependent ATPase, a 5′-phosphatase, and two proteins revealed by polyacrylamide gel electrophoresis. They also affect modification of host RNA polymerase, suppression of unwinding protein deficiency (sud phenotype), and ability to grow on two different Escherichia coli strains. The effect of different deletions on these properties demonstrates that at least seven genes are involved and allows their approximate locations to be determined. The general applicability of the method used for selecting deletions of nonessential DNA is discussed.

Recombination in bacteriophage λ: III. Studies on the nature of the prophage attachment region

Abstract The λ prophage attachment region (att) may be partially characterized by the results of vegetative phage crosses in which recombination is promoted by the site-specific prophage integration system (Int). This sort of recombination among λ+ and the variants λb2, λbio and λb2bio occurs at characteristic frequencies determined by both parents. Extensive control experiments strongly suggest that these frequency differences reflect structural differences among the att regions of the four phages, rather than functional differences in the Int system. It is therefore very likely that the att in phage λ is structurally different from the corresponding one in its host Escherichia coli. The results suggest that structural elements affecting Int-promoted recombination frequency lie on both sides of the Int-promoted crossover locus, and that elements on one side interact with those on the other. A crude model for att is discussed.

Characteristics of λp4, a λ derivative containing 9% excess DNA

Abstract In order to study virion morphogenesis in phage λ, we are synthesizing prophage genomes which are longer than normal and investigating what happens when they are induced. Our minor goal is to determine the upper limit to the quantity of DNA that can be incorporated into a phage head. Our major goal is to investigate DNA cutting, capsid formation, and DNA incorporation into the capsid when the genome is too long to be contained in the normal phage head. In the first application of this approach, we have constructed a prophage genome containing 9% excess DNA. Upon induction, this genome produces viable particles whose density and rate of heat inactivation indicate they contain the complete genome in a normal capsid. However, these phage have a reduced burst size which is attributable to the physical size of the genome.

A mutant of Escherichia coli that prevents growth of phage lambda and is bypassed by lambda mutants in a nonessential region of the genome.

Abstract A mutant strain of E. coli (rap − ) has been isolated which blocks the growth of phages λ, 434, and ∅80. Phage 424 and various non-lambdoid phages are not affected. λ mutants that can bypass the rap block are of two general types. First, a presumed point mutant mapping between h and att provides partial bypass of the block. Second, all deletions and substitutions that eliminate or substitute one or both recognition regions of the attachment site restore a high level of growth. However, a point mutation in the crossover region of the attachment site does not bypass the block, and the rap mutation does not affect Int-promoted λ recombination. While the mechanism by which rap blocks phage development is unknown, the block implies a new and potentially interesting form of phage-host interaction.

The nature and origin of a class of essential gene substitutions in bacteriophage λ

Abstract Particles containing extra DNA are found in high-titer stocks prepared by growth of the λ deletion mutants λb 221 and λb 221 imm434 (−21.9 and 24.1% λ DNA, respectively). We have found that stocks of λb 221 contain, in addition to the phage with tandem duplications previously reported (Emmons, S. W., MacCosham, V., and Baldwin, R. L. (1975). J. Mol. Biol. 91, 133–146), a low level of stable addition and substitution mutants that result in an increase in DNA content. The substitution mutants are present at a higher frequency in stocks of λb 221 imm434, where they are readily identified by their density in CsCl and their characteristic plaque morphology. Electron microscopic examination of heteroduplex DNA shows that the substitutions contain DNA related to the p 4 substitution. A number of strains of Escherichia coli K12 have been examined for their ability to yield this fragment of DNA. We find that λ with the p4 -like substitution was present in stocks of λb 221 imm434 grown on most strains but was absent in stocks grown on AB1157 or its derivatives. It has been suggested previously that the DNA present in the p 4 substitution might represent part of a cryptic prophage (Herskowitz, I., and Signer, E. R. (1974). Virology 61, 112–119). Our finding that the DNA is rescuable from many E. coli strains and that other fragments of the bacterial genome are not rescued supports this hypothesis. The absence of the cryptic fragment from AB1157 parallels the absence of other phenomena that have been attributed to a cryptic prophage, suggesting that the p 4 DNA may be part of a larger cryptic fragment.

Morphogenesis of bacteriophage lambda deletion mutants: I. Abnormal head-related structures produced in normal Escherichia coli

Abstract Late in the morphogenesis of bacteriophage lambda, DNA condenses into the nascent head and is cut from a concatemeric replicative intermediate by a nucleolytic function, Ter, acting at specific sites, called cos . As a result of this process, heads of lambda deletion mutants contain less DNA than those of the wild-type phage. It has been reported that phage with very large deletions (22% of the genome or more) grow poorly but that normal growth can be restored by the non-specific addition of DNA to the genome. This finding implies that DNA content may exert a physical effect on some stage of head assembly. We have investigated the effects of two long deletions, b 221 and tdel 33, on head assembly. Bacteria infected with the mutants were lysed with non-ionic detergent under conditions favoring stabilization of labile structures containing condensed DNA. It has proved possible to isolate two aberrant head-related structures produced by the deletion mutants. One of these (“overfilled heads”) contains DNA which is longer than the deletion mutant genome and is about the same size as that found in wild-type heads. These structures appear to be unable to attach tails. The second type of structure (“incompletely filled heads”) contains a short piece of DNA, 40% of the length of the mutant genome. The incompletely filled heads are found both with and without attached tails. Both of these abnormal structures are initially attached to the replicating DNA but are released by treatment with DNAase. The nature of these abnormal structures indicates that very small genomes affect a late stage of head morphogenesis, after the DNA is complexed with a capsid of normal size. The results presented suggest that underfilling of the capsid interferes with the ability of the Ter function to properly cleave cos .

Morphogenesis of λ with genomes containing excess DNA: Functional particles containing 12 and 15% excess DNA

Abstract In order to probe the mechanism of λ head morphogenesis, we have synthesized prophages whose genomes contain extra DNA and are investigating the processes of morphogenesis when they are induced. With genomes containing 12% or more excess DNA, the majority of particles produced are apparently nonfunctional or unstable. We report here the properties of the rare, stable, functional particles which are produced. Genomes of size 112% produce three types of functional particles: (1) particles containing a shorter genome due to a deletion mutation (burst size = 10 −3 ), (2) particles containing the complete genome in an abnormal capsid (burst = 10 −2 ) and (3) particles containing the complete genome in a normal capsid (burst = 10 −2 ). Genomes of size 115% produce only the former two types, each at about ten-fold lower frequency. The particles with abnormal capsids have low, heterogeneous density in CsCl gradients but normal sedimentation rate. They appear similar to the phenotypically abnormal particles of λ wild type which are resistant to inactivation by chelating agents (one class of λ∗ particles, Parkinson and Huskey, 1971 ). Their resistance to inactivation and ability to encapsulate extralong genomes suggests they may have a role in nonlaboratory, real-life situations.

Morphogenesis of bacteriophage lambda deletion mutants: II. Escherichia coli mutants which prevent maturation of short genomes☆

Abstract We have isolated mutants of Escherichia coli which severely reduce the growth of bacteriophage lambda carrying the b221 deletion. Some of the bacterial strains also cause a moderate reduction in the growth of wild-type phage. In the mutant hosts tested, the growth of λ b 221 is restored by chromosomal alterations producing a non-specific increase in genome length. Thus the defect in growth can be attributed to the physical size of the genome, rather than a genetic effect of the b221 deletion. Our experiments show that the failure to grow results from a block to head morphogenesis and that growth can be restored by mutations in at least two phage head genes. In the accompanying paper we have shown that even in the normal bacterium, the process of packing and cutting the λ b221 genome is perturbed as a result of its small size. The block to morphogenesis in the bacterial mutant we have studied most extensively appears to result from an enhancement of the same effect. The experiments described support the hypothesis that there is host participation in the cutting of encapsulated lambda DNA, although it is not yet clear if this involves the direct participation of a host gene product.

chi Mutations of Phage Lambda

Results of experiments carried out in a number of laboratories demonstrate the interdependence of replication, recombination, and maturation in phage lambda (see, for example, the review by Stahl et al., 1973, and references cited there). The experiments summarized in this report relate to the role of recombination promoted by the Escherichia coli Rec system in the growth of deletion mutants of lambda that have lost the genes between att and cIII (Fig. 1). A detailed description of this work will be presented elsewhere (Henderson and Weil, manuscript in preparation).