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Previous work identified gene product 56 (gp56), encoded by the lytic bacteriophage SP01, as being responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here, we show that expression of the predicted 9.3-kDa gp56 of SP01 inhibits later stages of B. subtilis cell division without altering FtsZ ring assembly. Green fluorescent protein-tagged gp56 localizes to the membrane at the site of division. While its localization does not interfere with recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analyses suggest that gp56 localization and activity depend on its interaction with FtsL. Together, these data support a model in which gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis. IMPORTANCE Studies over the past decades have identified bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. The phage factors causing cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanisms of several phage factors that inhibit cytokinesis, including gp56 of bacteriophage SP01 of Bacillus subtilis, remain unexplored. Here, we show that, unlike other published examples of phage inhibition of cytokinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and to block recruitment of proteins needed for septal cell wall synthesis.

Previous work identified gp56, encoded by the lytic bacteriophage SP01, as responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here we show that expression of the predicted 9.3-kDa gene product 56 (gp56) of SP01 inhibits latter stages of B. subtilis cell division without altering FtsZ ring assembly. GFP-tagged gp56 localizes to the membrane at the site of division. While its localization permits recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analysis suggest that gp56 localization and activity depends on its interaction with mid-recruited proteins DivIC and/or FtsL. Together these data support a model where gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCEResearch over the past decades has uncovered bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. Phage factors that cause cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanism of several identified phage factors that inhibit cytokinesis remain unexplored, including gp56 of bacteriophage SP01 of Bacillus subtilis. Here, we show that unlike related published examples of phage inhibition of cyotkinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and block recruitment of proteins needed for the septal cell wall synthesis.

This chapter describes the procedure that we have used to introduce suppressible nonsense mutations into various genes of Bacillus subtilis bacteriophage SPO1. The targeted gene is cloned in a B. subtilis/Escherichia coli shuttle vector. Using an in vitro enzymatic procedure dependent on mutant oligonucleotide primers, a mutation is inserted into the cloned gene, replacing an early lysine codon (AAA or AAG) with a nonsense codon (TAG or TAA). The mutant plasmid is recovered by transformation into E. coli, and is then transformed into B. subtilis carrying a suppressor that inserts lysine at TAG or TAA codons. Recombination is allowed between the mutant plasmid and superinfecting wild-type SPO1, and mutant progeny phage are identified by plaque-lift hybridization to labeled oligonucleotides having the mutant sequence. This procedure is adaptable for other types of mutations, and for other phage-bacteria combinations for which appropriate strains and plasmids are available.

This chapter describes the procedure that we have used to introduce suppressible nonsense mutations into various genes of Bacillus subtilis bacteriophage SPO1. The targeted gene is cloned in a B. subtilis/Escherichia coli shuttle vector. Using an in vitro enzymatic procedure dependent on mutant oligonucleotide primers, a mutation is inserted into the cloned gene, replacing an early lysine codon (AAA or AAG) with a nonsense codon (TAG or TAA). The mutant plasmid is recovered by transformation into E. coli, and is then transformed into B. subtilis carrying a suppressor that inserts lysine at TAG or TAA codons. Recombination is allowed between the mutant plasmid and superinfecting wild-type SPO1, and mutant progeny phage are identified by plaque-lift hybridization to labeled oligonucleotides having the mutant sequence. This procedure is adaptable for other types of mutations, and for other phage-bacteria combinations for which appropriate strains and plasmids are available.

When Bacillus subtilis is infected by bacteriophage SPO1, the phage directs the remodeling of the host cell, converting it into a factory for phage reproduction. Much synthesis of host DNA, RNA, and protein is shut off, and cell division is prevented. Here I describe the protocols by which we have demonstrated those processes, and identified the roles played by specific SPO1 gene products in causing those processes.

When Bacillus subtilis is infected by bacteriophage SPO1, the phage directs the remodeling of the host cell, converting it into a factory for phage reproduction. Much synthesis of host DNA, RNA, and protein is shut off, and cell division is prevented. Here I describe the protocols by which we have demonstrated those processes, and identified the roles played by specific SPO1 gene products in causing those processes.

SPOl, SP82, Fe, 2C, SP8, H1, and SP5C constitute a family of large, virulent bacteriophages of Bacillus subtilis whose distinguishing feature is the complete replacement of thymine by hydroxymethyluracil in their DNA. This chapter focuses on SPO1, about which the most is known, but will also discuss data for the other phages that can illuminate areas not studied with SPO1. Gene product 28 substitutes for the host sigma factor, changing the specificity of the RNA polymerase so that it now transcribes the middle genes, which include genes 33 and 34. The products of these genes then modify the RNA polymerase so that it transcribes the late genes. The gene 27 mutant is also deficient in DNA replication, and neither deficiency appears to be an indirect result of the other. Thus, gp27 may play a direct role in both replication and late transcription, although it remains possible that either deficiency is the result of a polar effect on a downstream gene. All major early promoters are located in the terminal redundancy, as determined either by binding RNA polymerase to specific restriction fragments or by the formation of ternary complexes of restriction fragments, RNA polymerase, and nascent RNA. Recombination is essential for transfection by the DNA of hydroxymethyluracil (hmUra) phages. As shown primarily for SP82, transfecting DNA is susceptible to a host nuclease that causes sufficient damage that recombination between several genomes is necessary to form a single intact genome.

Although cells of Bacillus subtilis continue to grow after being infected by bacteriophage SPO1, they do not undergo cell division. The product of SPO1 gene 56 is necessary and sufficient for this inhibition of cell division. GP56 inhibits cell division when expressed in uninfected B. subtilis, without preventing cell growth, DNA synthesis or chromosome segregation, ultimately causing filamentation and loss of viability. During infection, a gene 56 mutation prevents the inhibition of cell division that occurs in wild-type infection. Under the laboratory conditions used, the gene 56 mutation did not affect burst size, latent period, or other components of the host-takeover process.

Bacteriophage genes offer a potential resource for development of new antibiotics. Here, we identify at least six genes of Staphylococcus aureus phage Sb-1 that have bactericidal activity when expressed in Escherichia coli. Since the natural host is gram-positive, and E. coli is gram-negative, it is likely that a variety of quite different bacterial pathogens would be susceptible to each of these bactericidal activities, which therefore might serve as the basis for development of new wide-spectrum antibiotics. We show that two of these gene products target E. coli protein synthesis.

We report the genome sequence of Bacillus subtilis phage SPO1. The unique genome sequence is 132,562 bp long, and DNA packaged in the virion (the chromosome) has a 13,185-bp terminal redundancy, giving a total of 145,747 bp. We predict 204 protein-coding genes and 5 tRNA genes, and we correlate these findings with the extensive body of investigations of SPO1, including studies of the functions of the 61 previously defined genes and studies of the virion structure. Sixty-nine percent of the encoded proteins show no similarity to any previously known protein. We identify 107 probable transcription promoters; most are members of the promoter classes identified in earlier studies, but we also see a new class that has the same sequence as the host sigma K promoters. We find three genes encoding potential new transcription factors, one of which is a distant homologue of the host sigma factor K. We also identify 75 probable transcription terminator structures. Promoters and terminators are generally located between genes and together with earlier data give what appears to be a rather complete picture of how phage transcription is regulated. There are complete genome sequences available for five additional phages of Gram-positive hosts that are similar to SPO1 in genome size and in composition and organization of genes. Comparative analysis of SPO1 in the context of these other phages yields insights about SPO1 and the other phages that would not be apparent from the analysis of any one phage alone. These include assigning identities as well as probable functions for several specific genes and inferring evolutionary events in the phages' histories. The comparative analysis also allows us to put SPO1 into a phylogenetic context. We see a pattern similar to what has been noted in phage T4 and its relatives, in which there is minimal successful horizontal exchange of genes among a "core" set of genes that includes most of the virion structural genes and some genes of DNA metabolism, but there is extensive horizontal transfer of genes over the remainder of the genome. There is a correlation between genes in rapid evolutionary flux through these genomes and genes that are small.

We show that the products of SPO1 genes 44, 50, and 51 are required for the normal transition from early to middle gene expression during infection of Bacillus subtilis by bacteriophage SPO1; that they are also required for control of the shutoff of host DNA, RNA, and protein synthesis; and that their effects on host shutoff could be accounted for by their effects on the regulation of gene expression. These three gene products had four distinguishable effects in regulating SPO1 gene expression: (i) gp44-50-51 acted to restrain expression of all SPO1 genes tested, (ii) gp44 and/or gp50-51 caused additional specific repression of immediate-early genes, (iii) gp44 and/or gp50-51 stimulated expression of middle genes, and (iv) gp44 and/or gp50-51 stimulated expression of some delayed-early genes. Shutoff of immediate-early gene expression also required the activity of gp28, the middle-gene-specific sigma factor. Shutoff of host RNA and protein synthesis was accelerated by either the 44 − single mutant or the 50 − 51 − double mutant and more so by the 44 − 50 − 51 − triple mutant. Shutoff of host DNA synthesis was accelerated by the mutants early in infection but delayed by the 44 − 50 − 51 − triple mutant at later times. Although gp50 is a very small protein, consisting almost entirely of an apparent membrane-spanning domain, it contributed significantly to each activity tested. We identify SPO1 genes 41 to 51 and 53 to 60 as immediate-early genes; genes 27, 28, and 37 to 40 as delayed-early genes; and gene 52 as a middle gene.

Early in infection of Bacillus subtilis by bacteriophage SPO1, the synthesis of most host-specific macromolecules is replaced by the corresponding phage-specific biosyntheses. It is believed that this subversion of the host biosynthetic machinery is accomplished primarily by a cluster of early genes in the SPO1 terminal redundancy. Here we analyze the nucleotide sequence of this 11.5-kb “host-takover module,” which appears to be designed for particularly efficient expression. Promoters, ribosome-binding sites, and codon usage statistics all show characteristics known to be associated with efficient function in B. subtilis. The promoters and ribosome-binding sites have additional conserved features which are not characteristic of their host counterparts and which may be important for competition with host genes for the cellular biosynthetic machinery. The module includes 24 genes, tightly packed into 12 operons driven by the previously identified early promoters P E 1 to P E 12. The genes are smaller than average, with half of them having fewer than 100 codons. Most of their inferred products show little similarity to known proteins, although zinc finger, trans-membrane, and RNA polymerase-binding domains were identified. Transcription–termination and RNase III cleavage sites were found at appropriate locations.

A cloned rpoB gene, specifying an apparently mutant RNA polymerase beta subunit, protected Escherichia coli against the cytocidal effects of the E3 protein of bacteriophage SPO1, suggesting that RNA polymerase is the primary cellular target of the E3 protein. Two segments of the wild-type E. coli genome, one of which specifies a suppressor of dnaK mutations, and thus, possibly, a molecular chaperone, also provided protection when overexpressed, but wild-type rpoB did not.

Some of the early genes of Bacillus subtilis bacteriophage SPO1 were hypothesized to function in the shutoff of host biosyntheses. Two of these genes, e3 and e22, were cloned and sequenced. E22 showed no similarity to any known protein, while E3, a highly acidic protein, showed significant similarity only to other similarly acidic proteins. Each gene was immediately downstream of a very active early promoter. Each was expressed actively during the first few minutes of infection and was then rapidly shut off and its RNA rapidly degraded. An e3 nonsense mutation severely retarded the degradation of e3 RNA. Expression of a plasmid-borne e3 gene, in either B. subtilis or Escherichia coli, resulted in the inhibition of host DNA, RNA, and protein syntheses and prevented colony formation. However, the e3 nonsense mutation caused no measurable decrease in either burst size or host shutoff during infection and, in fact, caused an increased burst size at high multiplicities of infection. We suggest that e3 is one of several genes involved in host shutoff, that its function is dispensable both for host shutoff and for phage multiplication, and that its shutoff function is not entirely specific to host activities.

Many of the Xba I, Eco RI * , Kpn l, and Bgl II fragments of bacteriophage SPO1, accounting for about 65% of the genomic sequences, were cloned in Bacillus subtilis . Four of the Eco RI * fragments were specifically refractory to cloning in both Escherichia coli and B. subtilis , probably because of expression of deleterious genes carried on the SPO1 fragments. To permit complete identification of the regions cloned, the SPO1 restriction map has been extended to include the Xba l fragments and the previously unmapped Kpn I fragments. Markers for 26 of the 39 known genes have been located on specific cloned fragments, permitting more precise determination of the positions of most of the genes. One cloned SPO1 fragment was inhibitory to SPO1 development.

Plasmid pBD64, a vector which is useful for cloning inBacillis subtilis ( T. J. Gryczan, A. G. Shivakumar, and D. Dubnau (1980) ,J. Bacteriol.141, 246–253), has at least three substantial transcription units. Two of these include the singleEcoRl,XbaI, andBamHI sites, while the other includes the singleBglII site. Each of these transcripts was synthesized in the counter-clockwise direction, relative to the pBD64 restriction map. No transcripts were detected in the opposite direction. Infection by bacteriophage SPO1 caused a substantial decrease in each of these transcripts. No new pBD64 transcripts were detected during SPOT infection. Various SPO1 genes, cloned at several of these pBD64 sites, were tested for expression by observing their capacity to complement SPO1 mutants. Several middle and late genes were expressed substantially, regardless of the orientation in which the fragments were inserted. Since transcription from the vector could cause expression only in one orientation, this argues that the necessary transcription originated at SPO1 promoters, and, thus, that SPO1 middle and late promoters can be active in thymine-containing DNA.

The hydroxymethyluracil-containing DNA of B. subtilis phage SPO1 is cleaved only with low efficiency by restriction nuclease Eco RI. The efficiency is greatly increased by Eco RI ∗ conditions, but even under these conditions, the efficiency is less than with thymine-containing DNA. In contrast with their effect on thymine-containing DNA, Eco RI ∗ conditions do not appear to increase the number of sites on the SPO1 genome at which cleavage takes place. We describe the fragments produced by a (nearly) limit digest of SPO1 DNA with Eco RI ∗ and assign most of the known SPO1 cistrons to specific fragments.

Mutants affected in cistrons 21 and 32 of bacteriophage SPO1 are defective specifically in the initiation of DNA replication. Mutations in cistron 32 also specifically affect the termination of replication.

By superinfection experiments, we show that prereplicative modification of the SPO1 DNA molecule requires, at most, a small fraction of the time between injection of the DNA and initiation of replication. Therefore, the timing of initiation of replication is controlled, primarily, by the time required for synthesis (and assembly) of the cellular machinery required for SPO1 replication.

Six children of similar ethnic origin with congenital myopathy, cleft palate, malignant hyperthermia (or susceptibility to malignant hyperthermia), and skeletal anomalies are presented. The findings are remarkably consistent among our patients, 3 of whom were related. This syndrome is likely to be inherited as an autosomal recessive trait. Children with this disorder are likely to undergo surgery with general anesthesia for facial or skeletal deformities and should be recognized as predisposed to developing malignant hyperthermia.

Mutations in 2 of the 36 cistrons known for Bacillus subtilis phage SPOT were unmappable because they showed high frequencies of recombination with each other and with mutations in all other cistrons (Okubo, S., Yanagida, T., Fujta, D. J., and Ohlsson-Wilhelm, B. M., 1972, Biken J. 15 , 81–97). These cistrons (35 and 36) are among the last to be replicated and their replication is specifically inhibited by mutants affected in SPOT cistron 32 (Glassberg, J., Franck, M., and Stewart, C. R., 1977a, J. Virol. 21 , 147–152). We propose that these observations can be explained by the location of these two cistrons within a region of terminal redundancy, and we present the following data in support of this proposal: (1) Mixed infection between sus 35 + and sus 35 − phage yields bursts which include a high proportion of heterozygotes. (2) The sus 35 genetic marker is located on two different restriction fragments. (3) By restriction nuclease mapping, one copy of this marker has been located to the right of the rightmost markers on the known genetic map. (4) Two of the fragments produced by Eco RI ∗ digestion of mature DNA are not found in digests of intracellular DNA, and no new fragment is found, suggesting that these fragments are part of a region of terminal redundancy used for the formation of concatemers. One of these fragments carries the sus 35 marker.

We describe a temperature-sensitive mutation of Bacillus subtilis which appears to affect a host function necessary for late protein synthesis during infection by bacteriophage SP82. When cells carrying this mutation are infected by SP82 at the restrictive temperature, synthesis of the late proteins responsible for serum-blocking power and lytic activity is inhibited, while relatively normal synthesis of an early enzyme continues, even at late times.

Normal levels of lytic activity are elicited by SP82 infection of Bacillus subtilis even in the absence of detectable SP82 DNA synthesis. This is contrary to the regulatory patterns of the lysozymes that are known to be phage specific, but is consistent with the hypothesis that the SP82 lytic enzyme is host specific. Normal levels of lytic activity are not, however, sufficient for lysis in SP82 infection. An additional function is required, whose activity can be prevented by an SP82 mutation that blocks DNA synthesis. A host mutation is described which renders this function unnecessary. The strain carrying this mutation can be lysed by SP82 in the absence of detectable DNA synthesis.

Published in Petroleum Transactions, AIME, Volume 213, 1958, pages 121–126. Abstract Reservoir performance predictions based on laboratory core test data assume that fluid flow is laminar for the laboratory test. A study has been made to determine the validity of this assumption for laboratory tests on various types of porosity found in producing limestone formations. Data are presented which show that turbulence and slippage can occur during laboratory tests on heterogeneous-type porosity limestones, thus causing serious errors in measured single-phase permeabilities and two-phase relative permeability characteristics. In single-phase flow tests it is possible to eliminate turbulence and correct for slippage or to eliminate both factors by controlling test conditions. It is not always possible to control test conditions and thereby eliminate turbulence and slippage in two-phase flow tests. A correction method is presented which can be used to calculate the true two-phase laminar flow relative permeability characteristic even though turbulence and slippage exist. Introduction It is customary to make use of Darcy's law and modifications of this law, together with laboratory data on formation core samples to predict the performance of producing reservoirs. Such predictions are based on an assumption that fluid flow is in the laminar or streamline region for the laboratory test. It was the purpose of this investigation to determine the extent to which turbulent flow may occur in laboratory fluid flow tests on heterogeneous porosity limestones. Considering that turbulent flow conditions might exist in some laboratory fluid flow tests, additional emphasis was placed on the development of a method to correct for turbulence when laminar flow conditions could not be attained. Fluid Flow Concepts for Porous Media The Influence of Pore Geometry on Fluid Flow One of the more important factors influencing fluid flow in porous media is the geometry of the pore space which includes such characteristics of the pores as size, shape, distribution, roughness, uniformity, etc. In general, oil- and gas-producing formations can be divided into two broad types on the basis of pore geometry.

Extractable lytic activity in competent cells of Bacillus subtilis 168 was markedly increased after treatment with homologous or heterologous deoxyribonucleic acid (DNA). This increase was prevented by deoxyribonuclease, and did not occur with B. subtilis W23 or with noncompetent B. subtilis 168 cells, neither of which take up DNA. Although the deoxyribonuclease-sensitive step in DNA uptake was completed within 10 min, the increase in lytic activity did not begin until more than 30 min after the addition of DNA. The increase was prevented by any of several antibiotics. These results are discussed in relation to the mechanisms for the uptake of transforming DNA and the lysis of transfected cells.

In a Bacillus subtilis deoxyribonucleic acid (DNA) preparation, extracted and purified by the Marmur procedure, the DNA molecules carrying a particular marker are heterogeneous with respect to molecular weight, buoyant density, and thermal stability. This finding constitutes evidence against unique points of breakage during DNA isolation. The variation in buoyant density suggests a local compositional heterogeneity in the chromosomal region of certain markers. The variation in molecular weight provides an explanation for the results of certain transformation experiments that are otherwise poorly understood. An example of such a result is the observation that acridine orange increases the efficiency of differential thermal inactivation of markers. An explanation of this phenomenon is suggested by the demonstration that acridine orange can decrease the natural intramarker heterogeneity in melting behavior.

Acridine Orange, at appropriate intermediate concentrations, causes a substantial broadening of the thermal transitions of Bacillus subtilis DNA and of dAT. Experiments in which the two polymers are healed together show that the broadening is the result of the transfer of acridine orange molecules from denatured to native DNA molecules.

A nonsense mutation in SPO1 gene 40 prevented normal shutoff of both host DNA and host RNA synthesis, showing that gp40 is required for the normal occurrence of both shutoffs. A gene 39 nonsense mutation caused accelerated shutoff of both host DNA and host RNA synthesis (aided by a gene 38 nonsense mutation), showing that gp39 (aided by gp38) limits the rate at which both shutoffs occur. The 40− mutation suppressed the accelerative effects of the 39− and 38− mutations, showing that gp40 also plays an essential role in the accelerated shutoffs. To the best of our knowledge, proteins with the particular activities implied for gp39 and gp40 have not been identified in any other bacteriophage. SPO1 has at least three different mechanisms that have the effect of delaying the shutoff of host DNA and RNA synthesis.

In mixed infections with Bacillus subtilis phages SP82 and SP01, the SP82 genotype is predominant among the progeny. This predominance is determined by a specific region of the genome, the pos region, which apparently is located near genes 29 to 32 (by the SP01 numbering system). Recombination between SP82 and SP01 yields phage which have both the SP82 pos region and an SP01 mutation. This mutation then behaves in mixed infection as if it were part of an SP82 genome.

When bacteriophage SP02 infects a Bacillus subtilis culture during or shortly after transformation, the frequency of transformants among the resulting lysogens is greatly reduced relative to that in the uninfected culture. The effect can occur after the deoxyribonucleic acid has been taken up and covalently attached to recipient deoxyribonucleic acid.

By using density-transfer and marker-rescue techniques, we have been able to determine that: (1) SPOT has at least two origins of replication; (2) one of these origins is located about 8% from the right-hand end of the genetic map (in or near cistron 32) and replication proceeds from this origin unidirectionally to the left; (3) the extreme right-hand 8% of the genetic map is replicated from right to left; and (4) an origin of replication lies either in, or to the left of, the extreme left-hand 8% of the genetic map.

Fragments of the DNA of bacteriophage SPO1 can be cloned in Bacillus subtilis , the natural host for SPO1. Clones of the same fragment are either stable or unstable, depending on the vector used. SPO1 DNA contains hydroxymethyluracil instead of thymine. The cloned fragments do not contain hydroxymethyluracil, even when it is present in the cell as a consequence of SPOT infection. Yet the cloned SPOT DNA is active both in expression of its genetic information and in recombination with superinfecting SPOT DNA. SPO1 gene 17, which is probably a late gene, is expressed from the cloned fragment regardless of the orientation of insertion, suggesting that it is transcribed from its natural promoter.

This study was performed to evaluate an automatic method of motor unit action potential (MUAP) analysis developed in our laboratory. MUAPs were recorded from the biceps brachii muscle of 68 normal subjects and 122 patients with nerve or muscle disease. The values of mean MUAP durations from normal subjects obtained by automatic analysis were similar to those reported in the literature. However, the normal range of MUAP amplitude and the incidence of polyphasic MUAPs were much higher. Normal ranges of mean MUAP area, area/amplitude ratio, and the number of turns were also defined. Automatic analysis demonstrated an abnormality of at least one MUAP feature in 70% of patients. There was concordance between automated analysis and visual assessment of MUAPs in 76% of patients with neuropathy but in only 50% of patients with myopathy. The relationships between different MUAP features seen in neuropathy and myopathy are explained in physiologic terms.

During infection of Bacillus subtilis by bacteriophage SP82, substantial quantities of at least two different late proteins were synthesized, even in the absence of detectable phage DNA replication. The two proteins were the lytic enzyme (which is possibly a host specific enzyme) and the protein(s) responsible for serum blocking power (which is presumably phage specific). The absence of replication was assured by the use of an SP82 double mutant carrying a mutation in each of two cistrons required for replication, and was verified by density transfer and isotope incorporation experiments. Thus, it appears that phage DNA replication is not required for late protein synthesis during SP82 infection. Using a closely related phage, 2C, Pene and Marmur (1967) had shown that, when DNA replication was inhibited by mitomycin C, an early enzyme was synthesized normally, but the lytic enzyme was not. We have repeated this observation with SP82 and have shown that the mitomycin also prevents the appearance of serum blocking power, eliminating the possibility that the effect of mitomycin is specific to host proteins. However, we have also shown that, with a suitable preincubation, mitomycin can also inhibit early enzyme synthesis. We suggest that the differential effect of mitomycin on early and late protein synthesis is merely the result of the time after application of mitomycin at which the two groups of proteins are normally synthesized. Gage and Geiduschek (1971) showed that a replication defective mutant of SP01, another closely related phage, failed to synthesize one class of late RNA. We discuss possible ways by which our data can be reconciled with this result.

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LONG SHARP foreign bodies in the stomach are alarming to patients, their relatives, and their physicians. Of course, they are potentially dangerous but most of them pass through the gastrointestinal tract without difficulty. When lodged in the esophagus they should be removed, usually by esophagoscopy but occasionally, in difficult cases, by thoracotomy. 1-3 Once they have safely reached the stomach gastroscopic removal is more difficult, normal passage of the foreign body is more likely, and the best policy is, usually, watchful waiting under close hospital and x-ray supervision. Such a policy was successfully carried out in the following case, which is reported because of its general interest and for reassurance of physicians in other similar cases. A 15-year-old boy was admitted to the hospital 5½ hours after swallowing a 6.5 cm. (2.5 in.) corsage pin. He had been asymptomatic since the accident and was asymptomatic on admission. Physical examination