Helios Murialdo

Bacteriophage lambda preconnectors: Purification and structure

The morphogenesis of bacteriophage lambda proheads is under the control of the four phage genes B, C, Nu3 and E, and the two Escherichia coli genes groEL and groES . It has been shown previously that extracts prepared from cells infected with a lambda C-E- mutant accumulate a gpB polymer, which behaves as a biologically active intermediate in prohead assembly. This gpB activity has been called a preconnector , as it is probably a precursor to the head-tail connector. We now report the partial purification of biologically active preconnectors and the characterization of its structure. In the electron microscope, preconnectors appear as donut -like structures composed of several subunits displaying radial symmetry. Optical filtration of periodic arrays of preconnectors showed that the structure has 12-fold rotational symmetry. Side views of the preconnector reveal that it resembles an asymmetrical dumbell . This information has been used to construct a three-dimensional model of the preconnector . The implications of this structure for prohead shape and function, and for DNA packaging are discussed.

Analysis of cosmids using linearization by phage lambda terminase

A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage lambda terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage lambda (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.

Secretion of a λ2 immunoglobulin chain is prevented by a single amino acid substitution in its variable region

Abstract We have studied two derivatives of the IgA ( λ 2 ) secreting myeloma cell line MOPC315: MOPC315.26, which produces and secretes a λ 2 light chain, and MOPC315.37, which produces but does not secrete the λ 2 chain. It has been reported that the only alteration in the MOPC315-37 λ 2 chain is located in the variable region (Mosmann and Williamson, (1980) Cell 20, 283–292). In order to determine the nature of this alteration, we cloned the fragment of the chromosome containing the rearranged λ 2 gene from both the nonsecreting variant MOPC315-37 and the normal λ 2 -secreting parent MOPC315-26 and determined their nucleotide sequence. We found that the nucleotide sequences coding for the leader peptide and for the constant region of the λ 2 chain were identical in the secretor and nonsecretor. The sequences of the variable region differed at a single base pair corresponding to the first nucleotide in the codon for amino acid number 15. MOPC315-26 has a G in this position creating the codon GGT which codes for glycine, and MOPC315-37 has a C in this position creating the codon CGT which codes for arginine. Thus, we have demonstrated that a single amino acid substitution of a neutral amino acid, glycine, for a positively charged amino acid, arginine, results in the failure of a protein to be secreted.

Studies on an in vitro system for the packaging and maturation of phage λ DNA

Abstract Packaging and maturation of λ DNA in cell-free extracts is arbitrarily partitioned into two stages. In stage 1, exogenously added, immature concatemeric λ DNA interacts with the A-gene product (pA) and phage proheads. After this reaction has occurred, a second extract is added to initiate a different set of events that results in phage production (stage 2). The use of highly purified pA and purified proheads during stage 1 results in a marked reduction in the final phage yield. The deficit is largely corrected by the addition of extracts of uninfected cells in stage 1. This observation suggests that one or more host factors are required in the packaging reaction. The testing in vitro for λ-specific requirements in stage 1 reveals that Nu1-negative extracts prepared from λt16-infected cells are phenotypically prohead positive, but pA negative, in the assay. This result indicates that the t16 mutation is in a locus that specifies a gene product. Complementation in vitro between λt16- and λAam-infected cell extracts has not been accomplished as yet. Paradoxically, highly purified pA can support optimal phage production in conjunction with Nu1-negative stage 1 and stage 2 extracts. Stage 2 requires the action of the products of λ genes D, W, and FII. Successful complementation among pairs of stage 2-deficient extracts provides convenient assays for purifying these proteins.

Early intermediates in bacteriophage lambda prohead assembly. II. Identification of biologically active intermediates.

The morphogenesis of bacteriophage lambda proheads is under the control of the four phage genes B, C, Nu3, and E, as well as the E. coli genes groEL and groES. It has been previously shown that extracts prepared from cells infected with a lambda C-E- mutant accumulate biologically active gpB and gpNu3 (Murialdo, H., and Becker, A., J. Mol. Biol. 125, 57-74 (1978) ). To characterize the nature of these intermediates in prohead assembly, extracts prepared from these cells were fractionated by DEAE-cellulose chromatography as well as velocity sedimentation. Intermediates containing gpB were identified by SDS-polyacrylamide gel electrophoresis and by their ability to be assembled into biologically active proheads in vitro. The results indicate that the most abundant, biologically active intermediate (greater than 98% of the gpB activity) is a 25 S gpB-containing polymer. A second biologically active intermediate (about 1% of the total gpB activity) was identified as a gpB-gpgroEL complex.

Early intermediates in bacteriophage lambda prohead assembly.

Abstract The morphogenesis of phage λ proheads is under control of the four phage genes B, C, E, and Nu3 and the host cell gene groE. To determine if the assembly of the prohead proceeds via the formation of subassembly complexes, the proteins synthesized in E. coli following infection with λ phage were analyzed by analytical sedimentation in glycerol gradients. The phage-induced proteins were labeled in vivo by incorporation of 35S-labeled Met, and the protein composition of the fractions of the gradients was determined by electrophoresis in polyacrylamide gels followed by autoradiography. It was found that gpB sediments at positions corresponding to 25 S and 30 S, and that two polypeptides, derived from gpC, sediment as 30 S. The 25 S and 30 S complexes accumulate in λE−-infected cells, but no complexes are formed in λE−B−-infected cells. The formation of the 30 S complex, but not of the 25 S complex, is blocked in λE−C−-infected cells. A host-controlled band cosediments with gpB in the 25 S position, but not in the 30 S position. Using a recombinant λ phage carrying the host groE gene, the band cosedimenting with gpB was identified as the product of the groE gene. The groE gene product and gpB seem to form a complex since amber peptides of gpB (about 2 5 the size of the wild-type product) still cosediment with gpgroE in the 25 S position of the gradient. The formation of the 30 S complex is blocked, and the synthesis of the 25 S complex strongly inhibited, in groE missense mutant cells infected with λE− The results suggest that gpB and gpgroE interact at an early stage in prohead morphogenesis to form a 25 S complex which seems to be a precursor of a 30 S gpB- and gpC-containing complex. The existence of a gpB-gpC complex suggests that these two gene products interact directly. Since gpB is located in the head-tail junction of the phage, it seems highly likely that the polypeptides pX1 and pX2, derived from gpC, are also located at the head-tail junction.

Bacteriophage lambda DNA maturation. The functional relationships among the products of genes Nul, A and FI.

Abstract The FI gene of bacteriophage λ functions in head assembly, but its exact role is not well understood. FI mutants are leaky, producing between 0.1 and 0.5 viable particles per infected cell. In order to investigate the function of the FI product (gpFI) in vivo , mutants of λ were isolated that are able to grow in the absence of gpFI. These mutants, called fin (for FI independence) map in the region of gene Nul and the beginning of gene A . Proteins made in cells infected with the fin mutants were labelled with [ 35 S]methionine and analysed by polyacrylamide gel electrophoresis. In addition, the levels of activity of the A product were measured in the in vitro DNA packaging assay. As a result of these experiments, the fin mutants can be classified in two groups. Upon infection, fin mutants of one group selectively produce three to fivefold more gpA than do wild-type phage fin mutants of the second group do not overproduce any λ late gene product detectable by the autoradiographic technique. gpA overproducers can also be isolated by selecting for λ Aam Wam phages that can plate on a weak suII cell strain. The mutation responsible for this pseudoreversion is called A op and maps in the Nu1-A region. A op is also a fin mutation, since its presence in λ FI − enables it to plate on non-permissive hosts. Therefore, it seems that one condition sufficient for normal growth of FI − phage is the overproduction of gpA. The nature of the fin mutations that do not result in gpA overproduction is discussed.

Bacteriophage lambda DNA packaging: The product of the FI gene promotes the incorporation of the prohead to the DNA-terminase complex☆

Lambda DNA packaging in vitro can be examined in stages. In a first step, lambda DNA interacts with terminase to form a DNA-enzyme complex, called complex I. Upon addition of proheads, in a second step, a ternary complex, complex II, containing DNA, terminase and the prohead is formed. Finally, upon addition of the rest of the morphogenetic components, complete phages are assembled. We have investigated the effect of the FI gene product (gpFI) in these reactions and found that a stimulation in phage yield is observed when gpFI is included early in the reaction, at the time when DNA, terminase and proheads interact to form complex II. Measurements of complex II formation revealed that gpFI stimulated the rate of formation of this intermediate. gpFI was further shown to stimulate the addition of proheads to preformed complexes I to give complex II, but the protein did not stimulate complex I formation.

The overproduction of DNA terminase of coliphage lambda

An artificial operon containing the genes coding for the two subunits of lambda DNA terminase, Nul and A, has been constructed. Derivatives of plasmid pBR322 served as the cloning vehicles. The transcription is driven by the pL promoter of phage lambda, and translation of the terminase genes was made efficient by the replacement of the wild-type ribosome-binding sites for those of lambda genes cII and/or D. The operon also carries the oL operator, and this enables regulation of its expression by a thermosensitive repressor. The synthesis of genes Nul and A products is extremely efficient upon derepression. Within 40 min after induction of the operon, the two subunits comprise about 20% of the total cellular protein mass. Crude extracts prepared from these overproducing strains are at least 100 times more active than extracts prepared from induced lambda lysogens in both promotion of lambda DNA packaging and cosmid cleaving. The ability to produce highly concentrated terminase would enormously facilitate the study of its structure and mechanism of action. These extracts are also extremely useful in techniques such as lambda DNA packaging, cosmid mapping and cosmid linearization to improve efficiency of integration into mouse eggs.

A Genetic analysis of bacteriophage lambda prohead assembly in vitro

Abstract Phage lambda DNA can be packaged into proheads to generate full heads in vitro . Upon addition of tails and other gene products to the cell-free assembly system, infectious phage are formed. This system provides a convenient assay to measure biologically active proheads. Thus, it has been shown that biologically active proheads are not produced in wild-type cells infected with phage mutants in genes B , C , Nu3 and E , nor in groE mutant cells infected with wild-type phage. However, proheads can be assembled in vitro upon incubation of a mixture of λ Nu 3 − and λ E − infected-cell extracts. This complementation reaction has been analyzed using extracts prepared from wild-type or groE − cells infected with phage carrying amber mutations in one or two prohead genes. The conclusion from this analysis can be summarized as follows. 1. (1) Among all the possible combinations of extracts of cells infected with single amber mutants, the E − + Nu 3 − combination is the only one that gives substantial complementation in vitro . 2. (2) gpC † can be contributed by either member of the complementing pair of extracts. 3. (3) An E − B − extract will not complement a Nu 3 − extract. 4. (4) Active gpB can only be derived from a Nu 3 + extract, and/or active gpNu3 can only be derived from a B + extract. It is, therefore, probable that gpB and gpNu3 interact in vivo before gpE is polymerized. 5. (5) The lack of in vitro complementation exhibited by certain pairs of mutant extracts can be explained conveniently by postulating that essential gene products (such as gpE) are consumed in the assembly of abnormal capsid structures in vivo . 6. (6) The B − Nu 3 − extract (gpE and gpC donor) does not require the function of the host groE gene product to provide complementing activity. 7. (7) groE function is essential for the production of the complementing activity in E − C − extracts (gpB and gpNu3 donor). 8. (8) If gpB and gpNu3 are synthesized in the absence of wild-type groE function, the defect in complementing activity cannot be repaired by the addition of groE + cell extracts. It thus appears that groE functions very early during prohead assembly, prior to the polymerization of the coat protein gpE.