The phi29 early
mRNA's synthesized in infected Bacillus subtilis were studied by using sedimentation velocity analysis,
polyacrylamide gel electrophoresis, and hybridization of phi29
DNA fragments generated by the
restriction endonuclease Eco RI. Viral RNAs synthesized in vivo in the resence of
chloramphenicol were found to hybridize to
Eco RI-A, -C, and -D fragments, but not to
Eco RI-B and -E fragments, of the viral genome. Major early
mRNA sedimenting as 16S material in neutral
sucrose gradients was examined in detail. Radioactive phi29
RNA, purified by
sucrose gradient centrifugation, was hybridized to either the
Eco RI-A or
Eco RI-C
DNA fragment. The
RNA was eluted from the hybrids and then tested for complementary hybrid formation with
Eco RI-A and -C fragments.
RNA eluted from the
Eco RI-A fragment annealed only to the
Eco RI-A fragment and not to the
Eco RI-C fragment. Similarly,
RNA eluted from the
Eco RI-C fragment hybridized to the
Eco RI-C and -D fragments. Viral RNAs synthesized in vitro using B. subtilis
RNA polymerase hybridized to both
Eco RI-A and -C
DNA fragments. Furthermore,
RNA initiated with [gamma-(32)P]
GTP also hybridized to both
Eco RI-A and -C fragments. These results indicate that there are at least two efficient promotors for early transcription on the phi29 chromosome. In addition, a low-molecular-weight
RNA initiated with [gamma-(32)P]
ATP was found to hybridize exclusively with the
Eco RI-A fragment. Kinetic studies of phi29
mRNA synthesis during the lytic cycle have shown that viral RNAs hybridizable to the
Eco RI-A and -C fragments are synthesized immediately after phage
infection. On the other hand,
mRNA specific for the
Eco RI-B fragment was not synthesized for several minutes after phage
infection. Based on the results of the in vivo and in vitro transcription studies, a transcription map of the phi29 chromosome is proposed.