Bacteriophage T4 head morphogenesis: host DNA enzymes affect frequency of petite forms

Petite T4 phage particles have a shorter head than normal T4 phage and contain less DNA. They are not viable in single infections but are able to complement each other in multiply infected cells. Such particles normally make up 1 to 3% of T4 lysates. We show here that lysates of T4 grown on Escherichia coli H560 (end-A^?, pol-A^?) contain 33% of such petite particles. These particles are identical in physical and biological properties to those described previously, only their high frequency is abnormal. The frequency of petite particles in lysates grown on H560 is controlled by the presence or absence of the gene for DNA polymerase I (pol-A1) and apparently also a gene for endonuclease I (end-A). The involvement of these host DNA enzymes with T4 head morphology and DNA content indicates that DNA is directly involved in head morphogenesis. Such an involvement is incompatible with models of T4 head morphogenesis in which dimensionally stable, preformed empty heads are precursors of filled heads. The processing or repair of DNA apparently helps decide whether the assembly of T4 head subunits produces normal or petite heads.     

Bacteriophage T4 head morphogenesis : On the nature of gene 49-defective heads and their role as intermediates

Following infection under non-permissive conditions, T4 mutants defective in gene 49 accumulate structures which appear in the electron microscope to be empty phage heads. These structures are seen in extracts prepared under a variety of conditions, as well as in sections of the mutant-infected cells. The 49-defective heads (300 s) can be separated from phage particles (1000 s) by sedimentation through a sucrose gradient. A temperature-sensitive gene 49 mutant, tsC9, accumulates 300 s heads following infection at 41.5 °C, but can be “rescued” by a shift-down to 25 °C during the latter half of the latent period. Evidence from pulse-chase isotopic labeling experiments suggests that the 49-defective heads are intermediates in head formation. ¹⁴C-Labeled lysine, incorporated into the 300 s fraction at 41.5 °C, is rapidly and almost quantitatively transferred into the 1000 s phage particle fraction following a chase with an excess of unlabeled lysine and a shift to low temperature. The same result is observed when puromycin (200 μg/ml.) or chloramphenicol (200 μg/ml.) is added to the culture before temperature shift, suggesting that the inactive gene 49 product produced at high temperature becomes active at low temperature. In pulse-chase experiments carried out with wild-type T4-infected cells during the latter half of the latent period, the labeling kinetics of the 300 s and phage particle fractions support a precursor-product relationship. Conservation of the 300 s head structures during conversion to phage is demonstrated by ¹³C-¹⁵N density labeling of tsC9-infected cells at 41.5 °C followed by transfer to ¹²C-¹⁴N medium, shift to low temperature, isolation and lysis of the phage particles formed and centrifugation of the phage ghosts to equilibrium in CsCl solution.     

Dual Functions of Bacteriophage T4D Gene 28 Product: Structural Component of the Viral Tail Baseplate Central Plug and Cleavage Enzyme for Folyl Polyglutamates I. Identification of T4D Gene 28 Product in the Tail Plug

The T4D bacteriophage gene 28 product is a component of the central plug of the tail baseplate, as shown by the following two independent lines of evidence. (i) A highly sensitive method for radioactive labeling of only tail baseplate plug components was developed. These labeled plug components were incorporated by a complementation procedure into new phage particles and were analyzed by radioautography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three new structural proteins were found in addition to the three known tail plug proteins (i.e., gP29, gP27, and gP5). One of the three newly identified components had a molecular weight of 24,000 to 25,000 and appeared to be a product of T4D gene 28. (ii) Characterization of mutants of Escherichia coli bacteriophage T4D which produced altered gene 28 products also indicated that the gene 28 product was a viral tail component. T4D 28^ts phage particles produced at the permissive temperature had altered heat labilities compared with parent T4D particles. We isolated a single-step temperature revertant of T4D 28^ts and found that it produced phage particles which phenotypically resembled the original T4D particles. Since the properties of the phage baseplate components usually determine heat lability, these two changes in physical stability after two sequential single mutations in gene 28 supported the other evidence that the gene 28 product was a viral baseplate component. Also, compared with parent T4D particles, T4D 28^ts and T4D 28am viral particles adsorbed at different rates to various types of host cells. In addition, T4D 28^ts particles exhibited a different host range than parent T4D particles. This T4D mutant formed plaques with an extremely low efficiency on all E. coli K-12 strains tested. We found that although T4D 28^ts particles adsorbed rapidly and irreversibly to the E. coli K-12 strains, as judged by gene rescue experiments, these particles were not able to inject their DNA into the E. coli K-12 strains. On the other hand, the T4D 28^ts revertant had a plating efficiency on E. coli K-12 strains that was quite similar to the plating efficiency of the original parent, T4D. These properties of phage particles containing an altered gene 28 product supported the analytical finding that the gene 28 product is a structural component of the central plug of the T4D tail baseplate. They also indicated that this component plays a role in both host cell recognition and viral DNA injection.     

Production of infectious particles at the nonpermissive temperature by a temperature-sensitive mutant of bacteriophage SH-133 specific forPseudomonas facilis

The preliminary characterization of a unique temperature-sensitive (ts) mutant of bacteriophage SH-133, designatedts18, is reported. The mutant showed a substantial reduction in the ability to form plaques at the nonpermissive temperature (32°C) when compared with its plaqueforming ability at the permissive temperature (27°C). However, the supernatant fromts18-infected cells grown at 32°C exhibited significant infectivity when assayed at 27°C, which indicates that the reduced titer ofts18 at 32°C is not due to its inability to form phage particles at that temperature. Phage particles produced at 32°C, but not at 27°C, were thermolabile when tested at 32°C. The thermolability of phage yields from cells mixedly infected at 32°C with increasing wild-type/ts18 input ratios was independent of the quantity of wild-type gene product per cell. Thermostable phage particles were yielded byts18-infected cells that received short pulses of permissive temperature during the latter part of the latent period. These data indicate that the defect of the mutant is due to the production of a nonstructural assembly protein that misfunctions when viral maturation proceeds at the nonpermissive temperature.     

Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. II. Morphogenetic pathway

We have identified and characterized structural intermediates in phage P22 assembly. Three classes of particles can be isolated from P22-infected cells: 500 S full heads or phage, 170 S empty heads, and 240 S “proheads”. One or more of these classes are missing from cells infected with mutants defective in the genes for phage head assembly. By determining the protein composition of all classes of particles from wild type and mutant-infected cells, and examining the time-course of particle assembly, we have been able to define many steps in the pathway of P22 morphogenesis.In pulse-chase experiments, the earliest structural intermediate we find is a 240 S prohead; it contains two major protein species, the products of genes 5 and 8. Gene 5 protein (p5) is the major phage coat protein. Gene 8 protein is not found in mature phage. The proheads contain, in addition, four minor protein species, PI, P16, P20 and PX. Similar prohead structures accumulate in lysates made with mutants of three genes, 1, 2 and 3, which accumulate uncut DNA. The second intermediate, which we identify indirectly, is a newly filled (with DNA) head that breaks down on isolation to 170 S empty heads. This form contains no P8, but does contain five of the six protein species of complete heads. Such structures accumulate in lysates made with mutants of two genes, 10 and 26.Experiments with a temperature-sensitive mutant in gene 3 show that proheads from such 3^? infected cells are convertible to mature phage in vivo, with concomitant loss of P8. The molecules of P8 are not cleaved during this process and the data suggest that they may be re-used to form further proheads.Detailed examination of 8^? lysates revealed aberrant aggregates of P5. Since P8 is required for phage morphogenesis, but is removed from proheads during DNA encapsulation, we have termed it a scaffolding protein, though it may have DNA encapsulation functions as well.All the experimental observations of this and the accompanying paper can be accounted for by an assembly pathway, in which the scaffolding protein P8 complexes with the major coat protein P5 to form a properly dimensioned prohead. With the function of the products of genes 1, 2 and 3, the prohead encapsulates and cuts a headful of DNA from the concatemer. Coupled with this process is the exit of the P8 molecules, which may then recycle to form further proheads. The newly filled heads are then stabilized by the action of P26 and gene 10 product to give complete phage heads.     

Mechanism of head assembly and DNA encapsulation in Salmonella phage p22. I. Genes, proteins, structures and DNA maturation

The functions of ten known late genes are required for the intracellular assembly of infectious particles of the temperate Salmonella phage P22. The defective phenotypes of mutants in these genes have been characterized with respect to DNA metabolism and the appearance of phage-related structures in lysates of infected cells. In addition, proteins specified by eight of the ten late genes were identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis; all but two are found in the mature phage particle. We do not find cleavage of these proteins during morphogenesis.The mutants fall into two classes with respect to DNA maturation; cells infected with mutants of genes 5, 8, 1, 2 and 3 accumulate DNA as a rapidly sedimenting complex containing strands longer than mature phage length. 5^? and 8^? lysates contain few phage-related structures. Gene 5 specifies the major head structural protein; gene 8 specifies the major protein found in infected lysates but not in mature particles. 1^?, 2^? and 3^? lysates accumulate a single distinctive class of particle (“proheads”), which are spherical and not full of DNA, but which contain some internal material. Gene 1 protein is in the mature particle, gene 2 protein is not.Cells infected with mutants of the remaining five genes (10, 26, 16, 20 and 9) accumulate mature length DNA. 10^? and 26^? lysates accumulate empty phage heads, but examination of freshly lysed cells shows that many were initially full heads. These heads can be converted to viable phage by in vitro complementation in concentrated extracts. 16^? and 20^? lysates accumulate phage particles that appear normal but are non-infectious, and which cannot be rescued in vitro.From the mutant phenotypes we conclude that an intact prohead structure is required to mature the virus DNA (i.e. to cut the overlength DNA concatemer to the mature length). Apparently this cutting occurs as part of the encapsulation event.     

Dual Functions of Bacteriophage T4D Gene 28 Product: Structural Component of the Viral Tail Baseplate Central Plug and Cleavage Enzyme for Folyl Polyglutamates II. Folate Metabolism and Polyglutamate Cleavage Activity of Uninfected and Infected Escherichia coli Cells and Bacteriophage Particles

We investigated the role of the T4D bacteriophage gene 28 product in folate metabolism in infected Escherichia coli cells by using antifolate drugs and a newly devised assay for folyl polyglutamate cleavage activity. Preincubation of host E. coli cells with various sulfa drugs inhibited phage production by decreasing the burst size when the phage particles produced an altered gene 28 product (i.e., after infection under permissive conditions with T4D 28^ts or T4D am28). In addition, we found that another folate analog, pyrimethamine, also inhibited T4D 28^ts production and T4D 28am production, but this analog did not inhibit wild-type T4D production. A temperature-resistant revertant of T4D 28^ts was not sensitive to either sulfa drugs or pyrimethamine. We developed an assay to measure the enzymatic cleavage of folyl polyglutamates. The high-molecular-weight folyl polyglutamate substrate was isolated from E. coli B cells infected with T4D am28 in the presence of labeled glutamic acid and was characterized as a folate compound containing 12 to 14 labeled glutamate residues. Extracts of uninfected bacteria liberated glutamate residues from this substrate with a pH optimum of 8.4 to 8.5. Extracts of bacteriophage T4D-infected E. coli B cells exhibited an additional new folyl polyglutamate cleavage activity with a pH optimum of about 6.4 to 6.5, which was clearly distinguished from the preexisting activity in the uninfected host cells. This new activity was induced in E. coli B cells by infection with wild-type T4D and T4D amber mutants 29⁻, 26⁻, 27⁻, 51⁻, and 10⁻, but it was not induced under nonpermissive conditions by T4D am28 or by T4D 28^ts. Mutations in gene 28 affected the properties of the induced cleavage enzyme. Wild-type T4D-induced cleavage activity was not inhibited by pyrimethamine, whereas the T4D 28^ts activity induced at a permissive temperature was inhibited by this folate analog. Folyl polyglutamate cleavage activity characteristic of the activity induced in host cells by wild-type T4D or by T4D gene 28 mutants was also found in highly purified preparations of these phage ghost particles. The T4D-induced cleavage activity could be inhibited by antiserum prepared against highly purified phage baseplates. We concluded that T4D infection induced the formation of a new folyl polyglutamate cleavage enzyme and that this enzyme was coded for by T4D gene 28. Furthermore, since this gene product was a baseplate tail plug component which had both its antigenic sites and its catalytic sites exposed on the phage particle, it was apparent that this enzyme formed part of the distal surface of the phage baseplate central tail plug.     

Bacteriophage T4 Virion Baseplate Thymidylate Synthetase and Dihydrofolate Reductase

Additional evidence is presented that both the phage T4D-induced thymidylate synthetase (gp td) and the T4D-induced dihydrofolate reductase (gp frd) are baseplate structural components. With regard to phage td it has been found that: (i) low levels of thymidylate synthetase activity were present in highly purified preparations of T4D ghost particles produced after infection with td⁺, whereas particles produced after infection with td⁻ had no measurable enzymatic activity; (ii) a mutation of the T4D td gene from td^ts to td⁺ simultaneously produced a heat-stable thymidylate synthetase enzyme and heat-stable phage particles (it should be noted that the phage baseplate structure determines heat lability); (iii) a recombinant of two T4D mutants constructed containing both td^ts and frd^ts genes produced particles whose physical properties indicate that these two molecules physically interact in the baseplate. With regard to phage frd it has been found that two spontaneous revertants each of two different T4D frd^ts mutants to frd⁺ not only produced altered dihydrofolate reductases but also formed phage particles with heat sensitivities different from their parents. Properties of T4D particles produced after infection with parental T4D mutants presumed to have a deletion of the td gene and/or the frd gene indicate that these particles still retain some characteristics associated with the presence of both the td and the frd molecules. Furthermore, the particles produced by the deletion mutants have been found to be physically different from the parent particles.     

Genetic Control of Capsid Length in Bacteriophage T4 I. Isolation and Preliminary Description of Four New Mutants

Four new mutants are described whose phenotypic expression affects the length of the head of bacteriophage T4D. All mutants produce some phenotypically normal phage particles. Mutant pt21-34 also produces at least two size classes of phage particle which have heads that are shorter than normal. The other three mutants, ptg19-2, ptg19-80, and ptg191, produce, in addition to phages with normal and with shorter-than-normal heads, giant phages with heads from 1.5 to at least 10 times the normal length. All mutations are clustered near gene 23. Giant phage particles have the following properties: they are infectious and contain and inject multiple genomes as a single continuous bihelical DNA molecule of greater-than-unit length. Their frequency, relative to the total plaque-former population, increases late in the infectious cycle. They have a normal diameter, variable length, and a buoyant density range in CsCl from equal to slightly greater than that of normal phage. The arrangement of capsomers is visible in the capsids, which are composed of cleaved gene 23 protein.     

Maturation of the head of bacteriophage T4. I. DNA packaging events

Pulse-chase experiments in wild-type and mutant phage-infected cells provide evidence that the following particles called prohead I, II and III are successive precursors to the mature heads. The prohead I particles contain predominantly the precursor protein P23 and possibly P22 (mol. wt 31,000) and IP III (mol. wt 24,000) and have an s value of about 400 S. Concomitantly with the cleavage of most of P23 (mol. wt 55,000) to P231 (mol. wt 45,000), they are rapidly converted into prohead II particles which sediment with about 350 S. The prohead II particles contain, in addition to P231, the major constituents of the viral shella—a core consisting of proteins P22 and IP III. In cell lysates, prohead I and prohead II particles contain no DNA in a DNase-resistant form and are not bound to the replicative DNA. We cannot, however, positively rule out the possibility that these particles may have contained some DNA while in the cells.The prohead II particles are in turn converted into particles which sediment with about 550 S after DNase treatment (prohead III). During this conversion about 50% of normal DNA complement becomes packaged in a DNase-resistant form, and roughly 50% of the core proteins P22 and IP III are cleaved. In lysates the prohead III particles are attached to the replicative DNA. The prohead III particle appears to be the immediate precursor of the full mature head (1100 S). Cleavage of protein P22 to small polypeptides and conversion of IP III IP III1 are completed at this time. No precursor proteins are found in the full heads. Studies with various mutant phage showed that the prohead II to III conversion is blocked by mutations in genes 16 and 17 and that the conversion of the prohead III particles to the mature heads is blocked by mutations in gene 49. Cleavage of the head proteins, however, occurs normally in these mutant-infected cells. We conclude that the cleavage of the major component of the viral shell, P23, into P231 precedes the DNA packaging event, whereas cleavage of the core proteins P22 and IP III appears to be intimately linked to the DNA packaging event. Models relating the cleavage processes to DNA encapsulation are discussed.     

Mutations in the N Terminus of the ?X174 DNA Pilot Protein H Confer Defects in both Assembly and Host Cell Attachment

The øX174 DNA pilot protein H forms an oligomeric DNA-translocating tube during penetration. However, monomers are incorporated into 12 pentameric assembly intermediates, which become the capsid''s icosahedral vertices. The protein''s N terminus, a predicted transmembrane helix, is not represented in the crystal structure. To investigate its functions, a series of absolute and conditional lethal mutations were generated. The absolute lethal proteins, a deletion and a triple substitution, were efficiently incorporated into virus-like particles lacking infectivity. The conditional lethal mutants, bearing cold-sensitive (cs) and temperature-sensitive (ts) point mutations, were more amenable to further analyses. Viable particles containing the mutant protein can be generated at the permissive temperature and subsequently analyzed at the restrictive temperature. The characterized cs defect directly affected host cell attachment. In contrast, ts defects were manifested during morphogenesis. Particles synthesized at permissive temperature were indistinguishable from wild-type particles in their ability to recognize host cells and deliver DNA. One mutation conferred an atypical ts synthesis phenotype. Although the mutant protein was efficiently incorporated into virus-like particles at elevated temperature, the progeny appeared to be kinetically trapped in a temperature-independent, uninfectious state. Thus, substitutions in the N terminus can lead to H protein misincorporation, albeit at wild-type levels, and subsequently affect particle function. All mutants exhibited recessive phenotypes, i.e., rescued by the presence of the wild-type H protein. Thus, mixed H protein oligomers are functional during DNA delivery. Recessive and dominant phenotypes may temporally approximate H protein functions, occurring before or after oligomerization has gone to completion.     

Bacteriophage lambda FII gene protein: role in head assembly

The in vitro completion of bacteriophage lambda F_II^? heads to form phage can be used as an assay for the λ F_II gene protein. F_II protein activity is released from highly purified phage particles or phage heads by treatment with heat or denaturing agents. F_II protein was purified from isolated phage particles and from an extract of E^? infected cells in which it is not bound to any large structures. No differences in molecular weight (11,500), isoelectric point (4.75), electrophoretic mobility, or purification properties could be demonstrated between the F_II proteins from the two sources. Thus the polypeptide does not seem to be modified during assembly.Phage φ80 is closely related to λ. φ80 heads will join to φ80 tails in vitro but will not join to λ tails, though λ heads will join to either type of tail. Mixing experiments between F_II^? heads, tails, and F_II protein from λ or φ80 show that the specificity of head-tail joining is correlated with the source of the F_II protein and not with the source of the other head proteins. Thus, F_II protein is apparently responsible for this specificity of head-tail joining.     

Maturation of the head of bacteriophage T4. II. Head-related, aberrant tau-particles

Three somewhat different types of particle accumulate in cells infected with a phage carrying a mutation in gene 21 (in addition to the tubular variant (polyhead) of the head). The major type is the so-called τ-particle. These particles are very fragile, associated with the cell membrane, and have a sedimentation coefficient of about 420 S. They possess no DNA if isolated, and contain predominantly the precursor proteins P23, P24, P22 and the internal protein IP III, in addition to protein P20 and several proteins of unknown genetic origin.The remainder of the particles are partially or completely filled with DNA. The ratio of τ-particles to these partially or completely filled particles depends upon the particular mutant (in gene 21) phage used. In cells infected with a phage carrying the amber mutation (N90) in gene 21, about 10% of the precursor head protein P23 is cleaved to P231, and correspondingly about 10% of the particles are partially or completely filled with DNA. In cells infected with the temperature-sensitive mutant (N8) in gene 21, about 1% of the particles are fully or partially filled, and correspondingly about 1% of the P23 is cleaved to P231. In either case, the DNA-associated particles contain predominantly the cleaved proteins P231 and IP III1, and have none of the P22 and IP III found in τ-particles. This observation, and the correlation of the amount of partially or completely filled particles with the extent of the cleavage of P23 in the lysates, strongly suggest that cleavage of the head proteins is required for DNA packaging to occur.The τ-particles have properties similar to the so-called prohead I particles which we have isolated as intermediates in wild-type head assembly (preceding paper). However, temperature shift-down experiments, using several different phage carrying temperature-sensitive mutations in gene 21, indicate that the bulk of the τ-particles cannot be used for normal phage production.     

Host participation in bacteriophage lambda head assembly

Mutants of Escherichia coli, called groE, specifically block assembly of bacteriophage λ heads. When groE bacteria are infected by wild type λ, phage adsorption, DNA injection and replication, tail assembly, and cell lysis are all normal. No active heads are formed, however, and head related “monsters” are seen in lysates. These monsters are similar to the structures seen on infection of wild-type cells by phage defective in genes B or C.We have isolated mutants of λ which can overcome the block in groE hosts and have mapped these mutants. All groE mutations can be compensated for by mutation of phage gene E (hence the name groE). Gene E codes for the major structural subunit of the phage head. Some groE mutants, called groEB, can be compensated by mutation in either gene E or in gene B. Gene B is another head gene.During normal head assembly the protein encoded by phage head gene B or C appears to be converted to a lower molecular weight form, h3, which is found in phage. The appearance of h3 protein in fast sedimenting head related structures requires the host groE function.We suggest that the proteins encoded by phage genes E, B and C, and the bacterial component defined by groE mutations act together at an early stage in head assembly.     

The lambda head-tail joining reaction: purification, properties and structure of biologically active heads and tails

Procedures were developed to obtain biologically active lambda heads and tails at high purity with 20 to 40% recovery. Free heads, free tails and phage particles differ markedly in stability. Phage are stable in solutions containing Mg²⁺ but tails are not. The protein subunits which form the shaft of the tail dissociate in the presence of Mg²⁺ and form multisubunit spherical structures. EDTA protects free tails against inactivation but disrupts heads and phage particles. The four carbon diamine, putrescine, stabilizes heads against inactivation; the three and five carbon diamines are less effective. Electron micrographs reveal a new “knob” structure at the distal end of the tail fiber of phage and of free tails. Tails released from EDTA-disrupted phage possess a “head-tail connector”, a structure not present on the tail before its joining with a head.     

Structural studies of bacteriophage lambda heads and proheads by small angle X-ray diffraction.

We have examined a series of lambda proheads and mature structures by small angle X-ray diffraction. This technique yields spherically averaged density distributions and some information about surface organization of particles in solution.We find that gpE ² of proheads and heads forms shells with one of two radii; A^?, B^?, groE^?, and Nu3^? proheads have shells of radius 246 Å, while mature heads, urea-treated A^? proheads and C^? proheads have a radius of 300 Å. The expansion of proheads to mature heads is accompanied by a corresponding decrease in the thickness of the shell. groE^? proheads contain a core. This core is lost spontaneously from the structure and is only observed if the structures are fixed with glutaraldehyde prior to examination by X-ray diffraction or electron microscopy.C^? proheads expand to mature head size spontaneously. A preparation of C^? proheads which was fixed with glutaraldehyde at an early stage of the purification had the smaller, prohead radius. Unfixed particles from this preparation expanded to the mature head size after further purification and standing in the cold for several days. This result suggests that gpC may be involved in regulating head expansion.The radii of the protein shells of mature heads are identical for a series of phages that contain between 78% and 105% of the wild-type complement of DNA, and this radius is the same as that of proheads expanded in the absence of DNA. These results with phage lambda indicate that assembly of a double shell structure composed of coat and scaffolding protein, followed by expansion to a larger shell containing only coat protein is a general feature of the morphogenesis of dsDNA phages.     

A mutation which bypasses the requirement for p24 in bacteriophage T4 capsid morphogenesis

A mutation (byp24) affecting the N-terminal region of p23 will suppress the lethal effects of am and ts mutations in gene 24. In the presence of normal p24, the byp24 alteration causes a delay in the cleavage of capsid proteins and the assembly of a high percentage of isometric, short-headed particles; therefore, the byp24 mutation can affect the length of the T4 capsid. In the absence of p24, 24^?byp24 double mutants show a reduced rate of cleavage of capsid precursor proteins, and a reduced rate of virus assembly.Iminunoprecipitation with anti-p24 serum has shown the presence of both p24 and p24^c in wild-type phage particles. The 24^?byp24 particles contain no p24 or p24^c, as determined by immunoprecipitation, urea/acrylamide gel electrophoresis, and two-dimensional isoelectric focusing, urea/acrylamide gradient gel electrophoresis. They have a normal electron microscopic appearance, pH stability, and heat stability; but they are more resistant to osmotic shock than wild-type T4. We suggest that p24 normally functions in the initiation of phage T4 capsid protein cleavage reactions.     

Ung1p-mediated uracil-base excision repair in mitochondria is responsible for the petite formation in thymidylate deficient yeast

The budding yeast CDC21 gene, which encodes thymidylate synthase, is crucial in the thymidylate biosynthetic pathway. Early studies revealed that high frequency of petites were formed in heat-sensitive cdc21 mutants grown at the permissive temperature. However, the molecular mechanism involved in such petite formation is largely unknown. Here we used a yeast cdc21-1 mutant to demonstrate that the mutant cells accumulated dUMP in the mitochondrial genome. When UNG1 (encoding uracil-DNA glycosylase) was deleted from cdc21-1, we found that the ung1Δ cdc21-1 double mutant reduced frequency of petite formation to the level found in wild-type cells. We propose that the initiation of Ung1p-mediated base excision repair in the uracil-laden mitochondrial genome in a cdc21-1 mutant is responsible for the mitochondrial petite mutations.     

A minor pathway leading to plaque-forming particles in bacteriophage lambda. Studies on the function of gene D

Lysates of bacteriophage λ, mutant in the head gene D, contain a minor amount of defective particles which can be isolated and complemented to infective particles by adding purified gene D product. The defective particles contain DNA with a specific infectivity in the helper assay of about 10% of phage DNA. This DNA is firmly held in the capsid and a tail is attached. Although the particles adsorb to sensitive bacteria, the DNA is not injected. The complemented, infectious particles differ from normal phage by having a lower density. After growing in a permissive host, phage particles of normal density are produced. The implications of the ability of gene D protein to bind to otherwise complete particles as a last step are discussed.     

Assembly of Both the Head and Tail of Bacteriophage Mu Is Blocked in Escherichia coli groEL and groES Mutants

Like several other Escherichia coli bacteriophages, transposable phage Mu does not develop normally in groE hosts (M. Pato, M. Banerjee, L. Desmet, and A. Toussaint, J. Bacteriol. 169:5504–5509, 1987). We show here that lysates obtained upon induction of groE Mu lysogens contain free inactive tails and empty heads. GroEL and GroES are thus essential for the correct assembly of both Mu heads and Mu tails. Evidence is presented that groE mutations inhibit processing of the phage head protein gpH as well as the formation of a 25S complex suspected to be an early Mu head assembly intermediate.