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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.

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.