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The lytic action of lysozyme upon Bacillus subtilis walls was studied by following the disappearance of bacillary-colony-forming units and the appearance of L-colony-forming-units. The rapidity of cell wall removal by lysozyme fluctuated markedly during growth in a chemically defined medium, presumably because subtle changes in the cell wall were constantly occurring. When lysozyme-sensitive bacilli were grown with chloramphenicol 10 pg./ml. for 3 hr they showed a notable increase in lysozyme resistance; at the same time, their walls almost doubled in thickness. As lysozyme attack proceeded in a given culture, the bacilli passed first through a rod-shaped osmotically sensitive stage, and then a spherical stage characterized by incomplete removal of cell wall before finally reaching the naked protoplast stage. The spherical forms with adherent wall residues formed L colonies on a medium containing the reversion inhibitor D-methionine and bacillary colonies on the same medium without D-methionine. Under the latter conditions, the cell wall residue served as a starting point for rebuilding of complete wall, much as residual wall permits reversion of Gram-negative spheroplasts to the bacillary state. In the presence of Dmethionine, the feedback sequence required for wall formation was severed, resulting in heritable propagation of the protoplast state.

SUMMARYThe lytic cycle of Bacillus subtilis phage 4IC required the presence of at least to raM-calcium. In the absence of this ion, the plaquing efficiency of the virus was reduced to less than o-I. Likewise, replacement of Ca 2+ with other divalent ions (Ba 2+, Sr ~+, Mg ~+, Mn 2+) resulted in reduced efficiencies.Adsorption of 4rc was Ca2+-dependent, requiring concentrations ranging from o't to To mu. Although more than 9o% of the phage adsorbed at o.[ mM-Ca 2+, successful infection could only be achieved at higher Ca 2+ levels. Sub-optimal concentrations of the ion resulted in the loss of 9o% of infected centres within I rain after the initiation of infection, indicating an early post-adsorption ion requirement. Penetration experiments with 32P-labelled phage DNA indicated that an irreversible inhibition of injection was occurring in the majority of the phage-bacterium complexes. A third level of cation involvement became apparent when phage-bacterium complexes in which penetration had occurred exhibited a greatly reduced burst size. The post-penetration ionic requirement occurred early in the infection process since dilution of infected complexes into Ca2+-free medium at 2"5 min p.i. resulted in reduced phage yields. The requirement was dispensable after 6 rain p.i., since infected complexes diluted into Ca~+-free medium at this time exhibited a normal one-step growth curve. Analysis of messenger RNA production by molecular DNA-RNA hybridization techniques indicated that transcriptional events were similar in the presence and absence of Ca ~+. At present, the identification of the third ion-dependent stage is unresolved.

Seven major histocompatibility (B) complex recombinants were evaluated for anti-Rous sarcoma response. In experiment 1, the BR5(F21-G19) recombinant haplotype both homozygous and in heterozygous combinations with B" and BZ1 haplotypes were compared to BlglBlg and BZ1/Bz1 chickens to determine the relative influence of the BF versus BG chromosomal segments on regression of Rous sarcoma virus-induced tumours. In experiment 2, six recombinant haplotypes BR3 (F2-G2 3), BR4( F2-G2 3), BR"( F2 1-G2 3) andBR8(F2-G2a,23) present in chickens heterozygous for normal haplotypes B", BZ3 or B2" were compared for anti-sarcoma response. A total of 1328 chickens were blood typed for B alloantigens at 17 days of age, inoculated in the wingweb with Rous sarcoma virus at 6 weeks and monitored for anti-tumour immune response over a 10-week period. Genotypes which shared the same BF haplotype, but differed in their BG regions, had similar anti-tumour responses, implicating the BF but not the BG region in tumour regression. Chickens carrying BF2 or BF2' had a strong anti-tumour response, while BFZ4 conferred a weaker response, regardless of the accompanying normal haplotype.All chickens were typed for B alloantigens at approximately 17 days of age using a selected

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