Mouse IgG subclasses display a hierarchy of in vivo activities, with IgG2a and IgG2b showing the greatest protective and pathogenic properties. These enhanced activities result, in part, from their ability to bind to a novel, gamma chain-dependent, activating IgG Fc receptor, FcgammaRIV. FcgammaRIV maps in the 75 kb genomic interval between FcgammaRII and FcgammaRIII; its expression is restricted to myeloid lineage cells, and it binds to IgG2a and IgG2b with intermediate affinity. No binding to IgG1 or IgG3 was observed. Blocking FcgammaRIV binding to pathogenic anti-platelet antibodies is sufficient to protect mice from antibody-induced thrombocytopenia. Thus, the FcgammaR system has evolved distinct activation receptors displaying selectivity for IgG subclasses, with IgG1 antibodies exclusively dependent on FcgammaRIII, whereas IgG2a and IgG2b show preferential dependence on FcgammaRIV activation. These distinct binding affinities for the IgG subclasses to FcgammaRs account for their differential protective and pathogenic activities in vivo.

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The molecular cloning and the determination of the nucleotide sequence of the ispA gene responsible for farnesyl diphosphate (FPP) synthase [EC 2.5.1.1] activity in Escherichia coli are described. E. coli ispA strains have temperature-sensitive FPP synthase, and the defective gene is located at about min 10 on the chromosome. The wild-type ispA gene was subcloned from a lambda phage clone containing the chromosomal fragment around min 10, picked up from the aligned genomic library of Kohara et al. [Kohara, Y., Akiyama, K., & Isono, K. (1987) Cell 50, 495-508]. The cloned gene was identified as the ispA gene by the recovery and amplification of FPP synthase activity in an ispA strain. A 1,452-nucleotide sequence of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected molecular weights of 8,951 and 32,158, respectively. A part of the deduced amino acid sequence of ORF-2 showed similarity to the sequences of eucaryotic FPP synthases and of crtE product of a photosynthetic bacterium. The plasmid carrying ORF-2 downstream of the lac promoter complemented the defect of FPP synthase activity of the ispA mutant, showing that the product encoded by ORF-2 is the ispA product. The maxicell analysis indicated that a protein of molecular weight 36,000, approximately consistent with the molecular weight of the deduced ORF-2-encoded protein, is the gene product.

We constructed a null allele of the ftsI gene encoding penicillin-binding protein 3 of Escherichia coli. It caused blockage of septation and loss of viability when expression of an extrachromosomal copy of ftsI was repressed, providing a final proof that ftsI is an essential cell division gene. In order to complement this null allele, the ftsI gene cloned on a single-copy mini-F plasmid required a region 1.9 kb upstream, which was found to contain a promoter sequence that could direct expression of a promoterless lacZ gene on a mini-F plasmid. This promoter sequence lies at the beginning of the mra cluster in the 2 min region of the E. coli chromosome, a cluster of 16 genes which, except for the first 2, are known to be involved in cell division and cell envelope biosynthesis. Disruption of this promoter, named the mra promoter, on the chromosome by inserting the lac promoter led to cell lysis in the absence of a lac inducer. The defect was complemented by a plasmid carrying a chromosomal fragment ranging from the mra promoter to ftsW, the fifth gene downstream of ftsI, but not by a plasmid lacking ftsW. Although several potential promoter sequences in this region of the mra cluster have been reported, we conclude that the promoter identified in this study is required for the first nine genes of the cluster to be fully expressed.Many of the genes involved in the cell division process in Escherichia coli are found localized in a few clusters on the chromosome. The best characterized of the clusters is located at 2 min. It has been designated the mra (for murein A) cluster (11,36,41). It contains at least six cell division genes (fts genes), together with seven murein biosynthetic genes (mur genes, mraY, and ddl) and a lipopolysaccharide biosynthetic gene (envA) (see Fig. 1). Among them is the ftsI (also named pbpB) gene, which codes for penicillin-binding protein 3 (PBP 3). PBP 3 is a cytoplasmic membrane protein that functions as DD-transpeptidase for the formation of a septum of the murein sacculus and is a principal lethal target of ␤-lactam antibiotics (1,16,26). Analyses of thermosensitive cell division mutants showing thermolabile penicillin binding of PBP 3 and their revertants indicated that PBP 3 is an essential cell division component (48), although addition of sucrose suppressed the division defect but not the PBP 3 thermolability. The transpeptidase activity is carried by the C-terminal penicillin-binding domain of PBP 3, while the function of the N-terminal domain is still controversial (16,26,50,57).We initiated an attempt to create a new class of ftsI mutants in the hope of understanding the function of the N-terminal domain of PBP 3, especially in terms of its interaction with other cell division proteins, and realized that a null allele of the ftsI gene on the chromosome would be very useful to characterize mutant alleles newly created on plasmids. To eliminate a possible effect of copy number, we decided to clone the mutant alleles on single-copy mini-F vectors rather than on multicopy vectors. ...

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