Isolation of a cDNA clone for the acyl carrier protein-I of spinach

A 715 base pair cDNA clone coding for an acyl carrier protein (ACP) in spinach leaves has been isolated and characterized. The amino acid sequence indicated by the cDNA sequence closely matches the amino acid sequence of the ACP-I isoform. The presence of polyadenylation and DNA sequence coding for a precursor protein with a putative transit peptide, and the absence of hybridization between the cloned DNA and isolated spinach plastid DNA collectively show that the ACP-I gene is nuclear-encoded. The ACP-I cloned DNA did not cross-hybridize with mRNA from spinach tissues in which ACP-II has been found. Cross-hybridization with mRNA from tissues of Brassica campestris was either weak or undetectable. The cloning of an ACP-I gene represents an initial step in the molecular dissection of fatty acid synthetase in plants.     

Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts

Summary DNA from a bacterial plasmid containing the T-DNA border sequences of Agrobacterium tumefaciens was transferred into the nucleus or the cytoplasm of tobacco mesophyll protoplasts by microinjection. Following culture in hanging drops, some of these protoplasts produced calli containing the foreign DNA sequences. Evidence for the presence of the injected plasmid DNA in these calli was provided by Southern hybridization analysis. The results demonstrated that random portions of the bacterial plasmid were linked to plant DNA and that integration did not occur at the T-DNA borders present on the injected plasmid. The average number of integrated copies ranged from less than one to 1–2 per tobacco genome. The frequency of integration averaged 14% with intranuclear injections compared to 6% with cytoplasmic injections. With further refinement, the use of microinjection may allow the introduction of many different types of genetic elements into plants.     

Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. I. Mouse

Summary Mouse salivary glands and pancreases were stained with a battery of ten horseradish peroxidase-conjugated lectins. Lectin staining revealed striking differences in the structure of oligosaccharides of stored intracellular secretory glycoproteins and glycoconjugates associated with the surface of epithelial cells lining excretory ducts. The percentage of acinar cells containing terminal -N-acetylgalactosamine residues varied greatly in submandibular glands of 30 male mice, but all submandibular acinar cells contained oligosaccharides with terminal sialic acid and penultimate -galactose residues. The last named dimer was abundant in secretory glycoprotein of all mucous acinar cells in murine sublingual glands and an additional 20–50% of these cells in all glands contained terminalN-acetylglucosamine residues. In contrast, terminal -N-acetylgalactosamine was abundant in sublingual serous demilune secretions. Serous acinar cells in the exorbital lacrimal gland, posterior lingual gland, parotid gland and pancreas exhibited a staining pattern unique to each organ. In contrast, the apical cytoplasm and surface of striated duct epithelial cells in the submandibular, sublingual, parotid and exorbital lacrimal gland stained similarly. A comparison of staining with conjugated lectins reported biochemically to have very similar carbohydrate binding specificity has revealed some remarkable differences in their reactivity, suggesting different binding specificity for the same terminal sugars having different glycosidic linkages or with different penultimate sugar residues.     

Ultrastructural visualization of galactose in the glycoprotein of gastric surface cells with a peanut lectin conjugate

Summary The ultrastructural localization of peanut lectin-binding sites in the gastric surface epithelial cell has been studied using a horseradish peroxidase-labelled peanut lectin (PL-HRP) conjugate and other cytochemical techniques. The PL-HRP procedure has visualized glycoprotein with presumed terminal galactose residues in the apical plasmalemma and secreted mucins and has localized such glycoprotein selectively in the intermediate Golgi cisternaé situated between the saccules of the maturing face and those of the forming face of the Golgi stacks. Other cytoplasmic organelles, including the forming and stored secretory granules, did not reveal glycoprotein with terminal galactosyl residues. These results demonstrate the applicability of the PL-HRP labelling technique at the electron microscopical level to localize not only extra but also intracellular peanut lectin-reactive sites. The observations afford information concerning the possible site in the Golgi apparatus where galactose residues are added to the growing oligosaccharide side-chain of mucous glycoprotein.     

Ultrastructural localization of acidic glycoconjugates with the low iron diamine method

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.     

Concanavalin A-horseradish peroxidase bridge staining of alpha-1 glycoproteins separated by isoelectric focusing on polyacrylamide gel.

The Coomassie Blue protein stain and the periodic acid-Schiff stain for glycoproteins are compared to a new method of staining glycoproteins resolved electrophoretically. The method utilizes a Concanavalin A-horseradish peroxidase sequence to visualize selectively glycoproteins with terminal or internal mannose or terminal N-acetylglucosamine. The method applied to characterization of M and Z allele products of alpha-l-antitrypsins separated by isoelectric focusing of polyacrylamide gels slabs have revealed differences in carbohydrate content of various components that were previously undetected.     

Immune complex receptors on cell surface. I. Ultrastructural demonstration of macrophages.

A method is described for ultrastructural localization of immune complex receptors on the surface of viable peritoneal exudate cells. The technique entails incubation with a soluble complex of horseradish peroxidase (HRP) and specific antibody to HRP at 4 degrees C followed by exposure to diaminobenzidine and processing for electron microscopy. The bound immune complexes were evident as focal deposits of HRP reaction product, adhering closely to the external surface of macrophages with an uninterrupted periodicity varying between 30 and 120 nm. Following incubation with an insoluble immune complex containing a higher proportion of antibody, receptor sites stained frequently, but large aggregates adhered to the cells. Rinsing cells after staining with soluble complexes partially displaced the bound immune complexes. Fixation prior to exposure to immune complexes largely eliminated the binding capacity of the immune complex receptors.     

NMR studies of structure, hydrogen exchange, and main-chain dynamics in a disrupted-core mutant of thioredoxin.

Core-packing mutants of proteins often approach molten globule states, and hence may have attributes of folding intermediates. We have studied a core-packing mutant of thioredoxin, L78K, in which a leucine residue is substituted by lysine, using 15N heteronuclear two- and three-dimensional NMR. Chemical shift differences between the mutant and wild-type main-chain resonances reveal that structural changes caused by the mutation are localized within 12 A of the altered side chain. The majority of resonances are unchanged, as are many 1H-1H NOEs indicative of the main-chain fold, suggesting that the structure of L78K is largely similar to wild type. Hydrogen exchange studies reveal that residues comprising the central beta-sheet of both mutant and wild-type proteins constitute a local unfolding unit, but with the unfolding/folding equilibrium approximately 12 times larger in L78K. The dynamics of main-chain NH bonds in L78K were studied by 15N spin relaxation and compared with a previous study of wild type. Order parameters for angular motion of NH bonds in the mutant are on average lower than in wild type, suggesting greater spatial freedom on a rapid time scale, but may also be related to different rotational correlation times in the two proteins. There is also evidence of greater conformational exchange in the mutant. Differences between mutant and wild type in hydrogen exchange and main-chain dynamics are not confined to the vicinity of the mutation. We infer that mispacking of the protein core in one location affects local dynamics and stability throughout.     

Conserved sequence motifs, alignment, and secondary structure for the third domain of animal 12S rRNA

Secondary structure models are an important step for aligning sequences, understanding probabilities of nucleotide substitutions, and evaluating the reliability of phylogenetic reconstructions. A set of conserved sequence motifs is derived from comparative sequence analysis of 184 invertebrate and vertebrate taxa (including many taxa from the same genera, families, and orders) with reference to a secondary structure model for domain III of animal mitochondrial small subunit (12S) ribosomal RNA. A template is presented to assist with secondary structure drawing. Our model is similar to previous models but is more specific to mitochondrial DNA, fitting both invertebrate and vertebrate groups, including taxa with markedly different nucleotide compositions. The second half of the domain III sequence can be difficult to align precisely, even when secondary structure information is considered. This is especially true for comparisons of anciently diverged taxa, but well-conserved motifs assist in determining biologically meaningful alignments. Patterns of conservation and variability in both paired and unpaired regions make differential phylogenetic weighting in terms of "stems" and "loops" unsatisfactory. We emphasize looking carefully at the sequence data before and during analyses, and advocate the use of conserved motifs and other secondary structure information for assessing sequencing fidelity.