Small (30S) subunits of Escherichia coli ribosomes are composed of 21 proteins and a 1542-nucleotide 16S rRNA, whose secondary structure is divided into three domains. An in vitro transcript of the 3' domain of 16S rRNA (residues 923-1542), assembles efficiently with 30S ribosomal proteins to form a compact ribonucleoprotein (RNP) particle. Isolated particles examined under the electron microscope have a globular appearance, similar in size and shape to the head of the 30S ribosomal subunit. Two-dimensional gel analysis of the particles indicates the presence of proteins S3, S7, S9, S1O, S13, S14, and S19 and smaller amounts of S2, all of which have been localized to the head of the 30S subunit by immunoelectron microscopy and neutron diffraction and belong to the S7 assembly family. Interestingly, protein S4, which is believed to interact exclusively with the 5' domain, is also reproducibly found associated with the particles in significant amounts. Chemical probing of the RNA in the assembled particle reveals characteristic cleavage protection patterns, showing that the proteins assemble with the 3'-domain RNA similarly to the way in which they assemble with 16S rRNA, although some of the later steps of assembly appear to be incomplete. These results show that the 3' domain of 16S rRNA can indeed assemble independently of the rest of the 30S subunit into a particle that resembles its structure in the ribosome. In addition, the assembled particles are able to bind spectinomycin with an affinity comparable to that of 30S subunits.Small ( Brimacombe and co-workers containing S7, S9, S19, either S13 or S14, and fragments derived from the 3' half of 16S rRNA after partial ribonuclease digestion of 30S small ribosomal subunits (3, 4). This was extended by Zimmermann (5) to isolation of specific RNP complexes corresponding to the 5' and central domains and demonstration that the deproteinized RNA fragments from each of the three domains were able to reassemble with specific subsets of ribosomal proteins. Nomura and co-workers showed that there were two to three independent nucleation events for in vitro assembly of 30S ribosomal subunits, suggesting independent assembly of different domains (6). Assembly mapping results suggest that one such nucleation event could be the binding of protein S7 to 16S rRNA, upon which the assembly of S2, S3, S9, S10, S13, S14, and S19 depends (7 The experiments presented here show that a subfragment of 16S rRNA, corresponding to the 3' domain of 16S rRNA, assembles with this same group of eight ribosomal proteinsthe S7 family-into a compact, globular RNP particle that resembles the head of the 30S ribosomal subunit. At least some 3'-domain particles are able to bind the antibiotic spectinomycin with an affinity and specificity comparable to that of complete 30S subunits. MATERIALS AND METHODST7 Transcription of rRNAs. Full-length 16S rRNA was transcribed from Bsu36I-linearized pBS16S.RS, a derivative of pBS16S.10B (13) in which 21 extra nucleotides preceding the 5' end of 16S r...

The objectives of the present study were to develop an antibody probe to the porcine estrogen-dependent oviductal glycoproteins and to determine, by use of immunogold electron microscopy, whether these glycoproteins become associated with oviductal and uterine oocytes and early embryos. Polyclonal antibody, prepared using the M(r) 75,000-85,000 glycoprotein, separated from other proteins by two-dimensional SDS-PAGE, specifically recognized all three estrogen-dependent glycoproteins (acidic 75,000-85,000 M(r); acidic 100,000 M(r); basic 100,000 M(r)). In ampullary tissue collected from ovariectomized and estrogen-treated gilts and from gilts at Day 1 of estrus, gold particles were clustered over putative secretory granules restricted to the apical region of secretory epithelial cells. While follicular oocytes did not react with immunoreactive colloidal gold, oviductal and uterine unfertilized oocytes were found to be densely and uniformly labeled by colloidal gold throughout the zona pellucida, associated with flocculent material in the perivitelline space, and associated with microvilli and vitelline membrane. Similarly, in oviductal (1-4-cell) and unhatched uterine (4-cell/blastocyst) embryos, colloidal gold particles were distributed throughout the zona pellucida, heavily associated with flocculent material in the perivitelline space, and associated with the plasma membrane of the blastomeres. Immunoreactive colloidal gold remained detectable within Day 7 hatched uterine embryos, but not with embryos from later days. These results further support the proposal that porcine estrogen-dependent oviductal glycoproteins are released into the oviductal lumen, become associated with oviductal and uterine oocytes and early embryos, and are retained by oocytes and early embryos in the uterus.

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The alpha-peptide of the rat brain sodium channel of apparent molecular weight 260K has been purified to homogeneity in order to determine its structural and chemical properties. By negative-stain electron microscopy, the molecule morphology of the solubilized channel protein appears as a stack of disks or rouleaux whose dimensions are 40 A X 200 A. Measurement of the secondary structure by circular dichroism shows that the alpha-peptide is a conformationally flexible polypeptide that contains mostly beta-sheet and random-coil in mixed detergent-phospholipid micelles and folds into a conformation that has approximately 65% alpha-helix after reconstitution into phosphatidylcholine vesicles. Preparative polyacrylamide gel electrophoresis was used to obtain a chemically homogeneous peptide to analyze the amino acid and carbohydrate composition. The amino acid composition shows a reasonably high content of acidic amino acids with no striking excess of hydrophobic amino acids, while carbohydrate analyses show that carbohydrate is 31% by weight of the protein with sialic acid representing over 50% of the total carbohydrates. The high alpha-helical content, the amino acid composition, and the large carbohydrate mass are similar to those of the eel electroplax sodium channel and appear to be general features of the sodium channels which have been analyzed structurally and chemically to date.

Historically, methods used to identify Thiothrix spp. in environmental samples have been inadequate because isolation and identification procedures are time-consuming and often fail to separate Thiothrix spp. from other filamentous microorganisms. We described a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) procedure which was used to identify Thiothrix spp. in wastewater, artesian springs, groundwater, and underwater subterranean samples. The ELISA utilized monoclonal antibody T3511 to a species-specific carbohydrate epitope of Thiothrix spp. No cross-reactions were observed among non-Thiothrix strains consisting of 12 species and nine genera. In field trials, the ELISA identified 100% of 20 biochemically and cytologically confirmed Thiothrix spp.-containing samples with no false positives. Indirect immunofluorescent microscopy utilizing T3511 was effective for wastewater samples but not for those from natural spring water because of background fluorescence in the latter. In addition, electron micrographs of Thiothrix spp. labeled with T3511-biotin-anti-mouse antibody-gold showed that epitope T3511 was intracellular both in laboratory strains and environmental isolates. The minimum level of detection of the ELISA was 0.10 g/ml.