The RNA bacteriophages of E. coli specifically encapsidate a single copy of the viral genome in a protein shell composed mainly of 180 molecules of coat protein. Coat protein is also a translational repressor and shuts off viral replicase synthesis by interaction with a RNA stem-loop containing the replicase initiation codon. We wondered whether the translational operator also serves as the viral pac site, the signal which mediates the exclusive encapsidation of viral RNA by its interaction with coat protein. To test this idea we measured the ability of lacZ RNA fused to the translational operator to be incorporated into virus-like particles formed from coat protein expressed from a plasmid. The results indicate that the operator-lacZ RNA is indeed encapsidated and that nucleotide substitutions in the translational operator which reduce the tightness of the coat protein-operator interaction also reduce or abolish encapsidation of the hybrid RNA. When coat protein is expressed in excess compared to the operator-lacZ RNA, host RNAs are packaged as well. However, elevation of the level of operator-lacZ RNA relative to coat protein results in its selective encapsidation at the expense of cellular RNAs. Our results are consistent with the proposition that this single protein-RNA interaction accounts both for translational repression and viral genome encapsidation.

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Escherichia coli rnh mutants defective in RNase H activity display the features of previously described sdrA (stable DNA replication) and dasF (dnaA suppressor) mutants: (i) sustained DNA replication in the absence of protein synthesis, (ii) lack of requirement for dnaA protein and the origin of replication (oriC), and (iii) sensitivity of growth to a rich medium. Both the sdrA mutants (selected for continued DNA replication in the absence of protein synthesis) and the dasF mutants (selected as dnaA suppressors) are defective in RNase H activity, measured in vitro. Furthermore, a 760-basepair fragment containing the rnh+ structural gene complements the phenotype of each of the rnh, sdrA, and dasF mutants, indicative of a single gene. One function of RNase H in vivo is in the initiation of a cycle of DNA replication at oriC dependent on dnaA+. In keeping with these results, RNase H contributes to the specificity of dnaA protein-dependent replication initiated at oriC in a partially purified enzyme system. Replication of the Escherichia coli chromosome starts at a unique site (oriC) and proceeds bidirectionally (1-3). The DNA fragment containing oriC has been cloned and sequenced (4, 5). Subsequent analyses of the structure and in vivo function of the oriC region have disclosed that a 245-base-pair sequence is required and that within this region certain unique sequences surrounded by defined stretches of spacer sequences must be strictly maintained (6, 7).The initiation of replication at oriC is dependent on several gene products, particularly dnaA (for review, see ref. 8). The dnaA protein, purified to near homogeneity (9), is an essential component required at an early stage of the initiation reaction in an in vitro system which replicates oriC plasmids (10, 11) and binds a 9-base-pair sequence which appears four times in oriC. The dnaB and dnaC gene products, essential for ongoing replication, are also required during or shortly after initiation (12, 13). RNA polymerase has also been implicated by the inhibitory effect of rifampicin at this stage of replication (14) and by a dnaA suppressor identified with rpoB, the l3-subunit gene of RNA polymerase (15).The initiation of each new round of replication of the E. coli chromosome requires de novo protein synthesis (14,16,17). The biochemical nature of this requirement for protein synthesis is still unknown. Mutants, termed constitutive stable DNA replication (Sdrc) mutants, have been isolated that maintain DNA synthesis despite the absence of protein synthesis (stable DNA replication) (18,19). One of these mutant alleles (sdrA224) maps at 5 min between metD and proA (unpublished observations). The sdrA224 mutant tolerates transposon insertional inactivation of the dnaA gene or oriC deletion (20). A group of extragenic suppressor mutations (dasF) of dnaAts mutations has been located near proA (21). The dasF mutants exhibit the Sdrc phenotype (unpublished observations). These several results suggest the presence of an alternative initiation pathway, distinc...

Most human blood basophils respond to FcεRI cross-linking by releasing histamine and other inflammatory mediators. Basophils that do not degranulate after anti-IgE challenge, known as “nonreleaser” basophils, characteristically have no or barely detectable levels of the Syk tyrosine kinase. The true incidence of the nonreleaser phenotype, its relationship (if any) to allergic asthma, and its molecular mechanism are not well understood. In this study, we report statistical analyses of degranulation assays performed in 68 control and 61 asthmatic subjects that establish higher basal and anti-IgE-stimulated basophil degranulation among the asthmatics. Remarkably, 28% of the control group and 13% of the asthmatic group were nonreleasers for all or part of our 4-year long study and cycling between the releaser and nonreleaser phenotypes occurred at least once in blood basophils from 8 (of 8) asthmatic and 16 (of 23) control donors. Microarray analysis showed that basal gene expression was generally lower in nonreleaser than releaser basophils. In releaser cells, FcεRI cross-linking up-regulated >200 genes, including genes encoding receptors (the FcεRI α and β subunits, the histamine 4 receptor, the chemokine (C-C motif) receptor 1), signaling proteins (Lyn), chemokines (IL-8, RANTES, MIP-1α, and MIP-1β) and transcription factors (early growth response-1, early growth response-3, and AP-1). FcεRI cross-linking induced fewer, and quite distinct, transcriptional responses in nonreleaser cells. We conclude that “nonreleaser” and “cycler” basophils represent a distinct and reversible natural phenotype. Although histamine is more readily released from basophils isolated from asthmatics than controls, the presence of nonreleaser basophils does not rule out the diagnosis of asthma.