The structure of the chorion with its associated surface filaments has been examined in Oryzias latipes using several techniques, including scanning and transmission electron microscopy, enzymatic digestion, and sodium dodecylsulfate-polyacrylamide gel electrophoresis. The chorion of the recently fertilized egg was found to be organized into three zones: an outer, fuzzy electron-lucent zone that was continuous over the surface of filaments, a middle, homogeneous electron-dense zone, and an inner zone of ten to 12 horizontal, fibrous lamellae. Two topographically distinct types of filaments were found on the chorionic surface: nonattaching and attaching. Nonattaching filaments showed a regular spatial distribution over the chorion with an interfilament distance of about 60-70 microns. Attaching filaments originated from a localized portion of the chorion and united with those of neighboring eggs to anchor the egg cluster to the gonoduct of the female. Both nonattaching and attaching filaments were morphologically regionalized into basal and distal segments. Internally, nonattaching and attaching filaments were constructed of unbranched, packed tubules with an average outside diameter of approximately 19.5 and 18.8 nm, respectively. Using the attaching filament for further study, it was determined by rotational analysis (Markham et al., '63) that the wall of each tubule was a cylinder composed of 14 globular subunits. Two structural types of attaching filaments were identified. The type I attaching filament was similar in internal organization to the nonattaching filament and consisted of only tubules. The type II attaching filament, however, showed a highly osmiophilic, electron-dense bar surrounded by packed tubules. Tubules of attaching filaments of the adult were resistant to the action of Triton X-100 and colchicine, but sensitive to a 0.1% protease solution. However, colchicine-treated ovary tissue showed an absence and pattern of disorganization of tubules at the periphery of developing filaments. Solubilized attaching filament samples electrophoresed on 7.5% polyacrylamide-SDS gels were resolved into a pair of Coomassie-blue-positive bands that comigrated with purified porcine brain tubulin. The apparent molecular weight of the attaching filament polypeptide was determined to be approximately 55,000 daltons. These data suggest that the extracellular, tubular components of attaching filaments (as well as nonattaching filaments) are proteinaceous and show properties similar to those of cytoplasmic microtubules. Tubular precursor material was electron-dense and appeared to originate in the cisternae of the rough endoplasmic reticulum of ovarian foll

The phosphoenolpyruvate:sugar phospho- MATERIALS AND METHODS Growth of E. coil Strains. The strains of E. coli used in this study are listed in Table 1. The plasmid pDIA100 (10) encodes the gene for adenylate cyclase; the plasmids pEL06 (9) and pDS20 (11) encode genes for the PTS proteins HPr, enzyme I, and a fragment of enzyme iiiGIc; and plasmid pDS48 (11) encodes the gene for enzyme IIIGIc. The plasmid pEL06 resident in strain 600 results in an approximately 20-to 70-fold overexpression of enzyme I (9, 11). Strains were grown in LB medium (12) at 370C with aeration. For studies involving the localization of 83-galactosidase, cells were grown to midlogarithmic phase, at which point 5 mM isopropyl B-D-thiogalactopyranoside was added and the cultures were allowed to grow for another hour. Plasmids were maintained in the appropriate strains by including in the cultures ampicillin (30 ,Ag/ml), kanamycin (50 jug/ml), or tetracycline (10 kg/ml). Overnight cultures of the strains were diluted 1:10 with fresh medium and grown for 4 hr. The cells were collected by centrifugation (10,000 x g) and then washed with phosphate-buffered saline (PBS) at pH 7.2. The washed cells were resuspended in 0.5% glutaraldehyde/0.6% tannic acid in PBS for 30 min at room temperature (7,8). The fixed cells were washed once with PBS followed by several washes in 30% (vol/vol) glycerol in PBS or 2.3 M sucrose in PBS for cryoprotection (13). After the final wash, the wet pellet was thoroughly mixed with a Vortex mixer. Drops of the thick suspension were placed on cryomicrotome specimen pins and quickly frozen by plunging them into liquid nitrogen-cooled propane (14, 15). These specimens were stored in a liquid nitrogen reservoir for future sectioning.Enzyme I and Antibody Reagents. Enzyme I was purified from E. coli strain 599 (9) as described (16). Samples of the purified protein were used for production of rabbit polyclonal antibody by standard procedures.fi-Galactosidase. The f3-galactosidase activity of strains KL16 and 600 were measured by the method of Miller (17). The activities were strain KL16 = 185 Miller units and strain 600 = 69 Miller units. Rabbit antibody against E. coli ,3-galactosidase was from Cappell Laboratories.Immunoelectron Microscopy. Specimen pins were mounted on the holder of a Cryonova cryomicrotome (LKB) and 60-to 80-nm-thick sections were cut with a freshly broken glass knife at -98°C. Ribbons of dry sections were collected from the edge of the knife on freezing drops of sucrose (13); they were then transferred to Formvar-coated electron microscope grids, which were placed on liquefied gelatin. Immunolabeling of the sections for revealing antigenic sites and staining of the sections on the grids for generating contrast of the cellular structures were accomplished by floating the grids on drops of solutions placed in Petri dishes. The treatments were done in the following sequence (all solutions were made in PBS): 0.02 M glycine, preimmune serum (1: 1000 dilution), immune serum (1:1000 dilution), protein A...

A protein with a tetragonal pattern, defined as RS protein, was found on the wall surface of an alkaline phosphatase secretion-deficient mutant (NM 105) of Bacillus licheniformis 749/C. The protein was present on the wall surface of the exponential-growth-phase cells, but at the stationary growth phase it was overproduced and hypersecreted. This protein was precipitated to homogeneity from the culture fluid by 80% ammonium sulfate saturation and chilled acetone. The molecular mass of the protein was 98 kilodaltons, and it had a single subunit in a sodium dodecyl sulfate gel. Specific anti-RS antibody was generated in rabbits and used to immunolabel the RS protein on the cells at different growth phases. In early-exponential-growth-phase cells, the outside surface of the wail, the cytoplasm, and the inside surface of the cytoplasmic membrane were labeled.In stationary-growth-phase cells, the cytoplasm was poorly labeled, but the labeling on the outside surface of the wall was high. A B. licheniformis NM 105 gene library was made by using the lambda phage EMBL3. The RS protein expression from this gene library was detected by a modified autoradiographic procedure. One of the amplified RS protein-positive plaques (4213-1) containing recombinant DNA was chosen, and the restriction map of this DNA was prepared. The RS protein expressed in Escherichia coli NM 539 infected with 4213-1 recombinant phage had a lower molecular mass than the purified authentic RS protein. The 4.5-kilobase-pair (kbp) Sall-EcoRI fragment of the recombinant DNA was cloned in the shuttle plasmid pMK4 to construct pMK462, which was expressed in B. subtilis MI112 and produced the RS protein identical in molecular mass to the purified authentic RS protein. The