We have explored the Escherichia coli chromosome architecture by genetic dissection, using a site-specific recombination system that reveals the spatial proximity of distant DNA sites and records interactions. By analysing the percentages of recombination between pairs of sites scattered over the chromosome, we observed that DNA interactions were restricted to within subregions of the chromosome. The results indicated an organization into a ring composed of four macrodomains and two less-structured regions. Two of the macrodomains defined by recombination efficiency are similar to the Ter and Ori macrodomains observed by FISH. Two newly characterized macrodomains flank the Ter macrodomain and two lessstructured regions flank the Ori macrodomain. Also the interactions between sister chromatids are rare, suggesting that chromosome segregation quickly follows replication. These results reveal structural features that may be important for chromosome dynamics during the cell cycle.

Using a series of mutants of Paramecium tetraurelia, we demonstrate, for the first time, changes in the internal structure of the cell membrane, as revealed by freezefracture, that correspond to specific single gene mutations. On the plasma membrane of Paramecium circular arrays of particles mark the sites of attachment of the tips of the intracellular secretory organelles--trichocysts. In wild-type paramecia, where attached trichocysts can be expelled by exocytosis under various stimuli, the plasma membrane array is composed of a double outer ring of particles (300 nm in diameter) and inside the ring a central rosette (fusion rosette) of particles (75 nm in diameter). Mutant nd9, characterized by a thermosensitive ability to discharge trichocysts, shows the same organization in cells grown at the permissive temperature (18~ while in cells grown at the nonpermissive temperature (27~ the rosette is missing. In mutant tam 8, characterized by normal but unattached trichocysts, and in mutant tl, completely devoid of trichocysts, no rosette is formed and the outer rings always show a modified configuration called "parentheses," also found in wild-type and in nd9 (18~ cells. From this comparison between wild type and mutants, we conclude: (a) that the formation of parentheses is a primary differentiation of the plasma membrane, independent of the presence of trichocysts, while the secondary transformation of parentheses into circular arrays and the formation of the rosette are triggered by interaction between trichocysts and plasma membranes; and (b) that the formation of the rosette is a prerequisite for trichocyst exocytosis.All freeze-fractured natural membranes studied so far display a smooth background that probably represents regions of lipid bilayer (5, 7), on which "particles" corresponding to proteins or lipoprotein aggregates (9, 20) are usually found. These particles generally appear randomly distributed but various types of organized particle arrays have been described: for instance, the arrays involved in 126

The trichocysts of Paramecium tetraurelia consitute a favorable system for studying secretory processes because of the numerous available mutations that block, at various stages, the development of these secretory vesicles, their migration towards and interaction with the cell surface, and their exocytosis .Previous studies of several mutants provided information (a) on the assembly and function of the intramembranous particles arrays in the plasma membrane at trichocyst attachment sites, (b) on the autonomous motility of trichocysts, required for attachment to the cortex, and (c) on a diffusible cytoplasmic factor whose interaction with both trichocyst and plasma membrane is required for exocytosis to take place.We describe here the properties of four more mutants deficient in exocytosis ability, nd6, nd7, tam38, and tam6, which were analyzed by freeze-fracture, microinjection of trichocysts, and assay for repair of the mutational defect through cell-cell interaction during conjugation with wild-type cells. As well as providing confirmation of previous conclusions, our observations show that the mutations nd6 and tam6 (which display striking abnormalities in their plasma membrane particle arrays and are reparable through cell-cell contact but not by microinjection of cytoplasm) affect two distinct properties of the plasma membrane, whereas the other two mutations affect different properties of the trichocysts . Altogether, the mutants so far analyzed now provide a rather comprehensive view of the steps and functions involved in secretory processes in Paramecium and demonstrate that two steps of these processes, trichocyst attachment to the plasma membrane and exocytosis, depend upon specific properties of both the secretory vesicle and the plasma membrane .The trichocysts of Paramecium are secretory vesicles whose formation in the cytoplasm, migration to the cell cortex, interaction with the plasma membrane, and exocytosis are easily observable by light and electron microscopy and are also amenable to genetic dissection. A number of mutations are available that block trichocyst development and exocytosis at various stages . These mutations disclose different steps and functions that might not be suspected or identified in other systems. The trichocyst system offers two further advantages for studying secretory processes .First, in the region of contact between trichocyst and plasma membrane, organized arrays of intramembrane particles are visible on freeze-fracture replicas of both the plasma and the

Previous studies on exocytosis in Paramecium using mutants affecting trichocyst extrusion permitted us to analyze the assembly and function of three intramembrane particle arrays ("ring" and "rosette" in the plasma membrane, "annulus" in the trichocyst membrane) involved in the interaction between these two membranes .Using a conditional mutation, nd9, which blocks rosette assembly and prevents exocytosis at the nonpermissive temperature, we have analyzed the effect of temperature on the secretory capacity of nd9 cells . By combining several techniques (physiological studies, microinjections, inhibition of fatty acid synthesis, and freeze-fracture analysis) we demonstrate (a) that the product of the mutated allele nd9 is not thermolabile but that its activity is dependent upon temperature-induced changes in the membrane lipid composition and (b) that the product of the nd9 locus is a diffusible cytoplasmic component whose interaction with both plasma membrane and trichocyst membrane is required for rosette assembly and exocytosis .The data provide physiological evidence for the existence of a molecular complex(es) linking the two membranes and involved in the control of membrane fusion ; we discuss the possible nature and function of these links .For three main reasons, the trichocysts of Paramecium constitute a model system for analysis of the mechanisms of exocytosis and membrane fusion . Firstly, a number of mutations are available that block the trichocyst cycle at various stages (trichocyst development, attachment to the plasma membrane, or exocytosis) . Secondly, exocytosis can be triggered at will and observed under the J . CELL. BIOLOGY