ANTHER AND POLLEN DEVELOPMENT IN RICE (ORYZA SATIVA)

Investigations of the growth of anthers and ontogeny of pollen grains of Oryza sativa (rice) IR-30 were undertaken for the purpose of 1) providing a set of growth measurements and 2) describing stable cytological features of anther and pollen development. Correlations exist between elongation of the floret and growth parameters of the anther such as its length, width, fresh and dry weights and cytological stage of pollen development. In the early ontogeny of the anther, hypodermal archesporial initials divide periclinally to form primary parietal cells and primary sporogenous cells. Each of the latter divides twice mitotically to generate four microspore mother cells, which undergo meiosis. The anther wall is formed by anticlinal and periclinal divisions of the primary parietal cells as well as of cells surrounding the primary sporogenous cells. Subsequent cytological features in the development of anther and pollen grains of rice have much in common with anther and pollen developmental biology of other members of Gramineae.     

Extended repertoire of permissible peptide ligands for HLA-B*2702.

Recognition of self peptides bound to the class I major histocompatibility complex molecule HLA-B27 is thought to trigger proliferation of autoreactive T cells and result in autoimmune arthritic diseases. Previous work from other laboratories established that a predominant feature of endogenous peptides eluted from purified B27 is an arginine at position 2. We studied the binding of peptides containing both natural and unnatural amino acids by the subtype HLA-B*2702, with the goal of gaining insight into peptide binding by this B27 subtype that is associated with susceptibility to arthritic disease. A soluble from of B*2702 was depleted of endogenous peptides. We tested the binding of peptides substituted with cysteine, homocysteine, or an alpha-amino-epsilon-mercapto hexanoic acid side chain (Amh) instead of the naturally occurring arginine at position 2, to determine whether the peptide sulfhydryl residue could be covalently linked to cysteine 67 in the B*2702 binding cleft. Although none of the altered peptide sequences bound covalently to B*2702, the affinities of the homocysteine- and Amh-substituted peptides were close to that of the native peptide sequence. Substitutions at position 2 with other side chains, such as glutamine and methionine, also resulted in peptides that bound with only slightly reduced affinity. These results demonstrate that peptide side chains other than arginine at position 2 can be accomodated within the B*2702 peptide binding site with only minor reductions in affinity. This extended repertoire of permissible B27-binding peptides should be taken into account for a consideration of disease-associated peptide sequences.     

Altered physicochemical characteristics of polyethylene glycol linked beet stem oxalate oxidase

Oxalate oxidase (EC 1.2.3.4), obtained from the beet stem, was covalently linked to polyethylene glycol (PEG). Compared with native enzyme, the modified oxalate oxidase exhibited decreased electrophoretic mobility, increased storage stability, higher thermal stability, and resistance to heavy metal inactivation and proteolytic digestion. The chemical modification of oxalate oxidase with PEG also brought about a marked shift in its optimal pH, from pH 4.5 to 6.5, without altering its Michaelis constant (K(m)) significantly. These acquired properties of the immobilized oxalate oxidase render it suitable for possible applications in clinical, nutritional, and medical fields. (c) 1995 John Wiley & Sons, Inc.     

Studies on the chemical modification of potato (Solanum tuberosum) lectin and its effect on haemagglutinating activity

1. Modification of potato (Solanum tuberosum) lectin with acetic anhydride blocked 5.1 amino and 2.7 tyrosyl groups per molecule of lectin and decreased the haemagglutinating activity of the lectin. De-O-acetylation regenerated 2.0 of the tyrosyl groups and resulted in a recovery of activity. 2. Modification with citraconic anhydride or cyclohexane-1,2-dione did not greatly affect activity, although modification of amino and arginyl groups could be demonstrated. 3. Treatment with tetranitromethane nitrated 3.7 tyrosine residues per molecule of lectin with concomitant loss of activity. The presence of 0.1m-NN′N″-triacetylchitotriose (a potent inhibitor of the lectin) in the reaction medium protected all the tyrosyl residues from nitration and the lectin was fully active. 4. Modification of tryptophyl groups with 2-hydroxy-5-nitrobenzyl bromide and 2,3-dioxoindoline-5-sulphonic acid modified 0.9 and 2.6 residues per molecule of lectin respectively with a loss of activity in each case. Reaction of potato lectin with 2,3-dioxoindoline-5-sulphonic acid in the presence of inhibitor protected 2.4 residues of tryptophan from the reagent. Loss of haemagglutination activity was prevented under these conditions. 5. Reaction of carboxy groups, activated with carbodi-imide, with α-aminobutyric acid methyl ester led to the incorporation of 5.3 residues of the ester per molecule of lectin. Presence of inhibitor in this case, although protecting activity, did not prevent modification of carboxy groups; in fact an increase in the number of modified residues was seen. This effect could be imitated by performing the reaction in 8m-urea. In both cases the number of carboxy groups modified was close to the total number of free carboxy groups as determined by the method of Hoare & Koshland [(1967) J. Biol. Chem. 242, 2447–2453]. Guanidination of lysine residues after carboxy-group modification gave less homoarginine than did the unmodified lectin under the same conditions, suggesting the formation of intramolecular cross-links during carbodi-imide activation. 6. It is suggested from the results presented that amino, arginyl, methionyl, histidyl and carboxyl groups are not involved in the activity of the lectin and that tyrosyl and tryptophyl groups are very closely involved. These findings are similar to those reported for other proteins that bind N-acetylglucosamine oligomers and also fit the general trend in other lectins.     

Gene activity during germination of spores of the fern, Onoclea sensibilis: RNA and protein synthesis and the role of stored mRNA

Pattern of 3H-uridine incorporation into RNA of spores of Onoclea sensibilis imbibed in complete darkness (non-germinating conditions) and induced to germinate in red light was followed by oligo-dT cellulose chromatography, gel electrophoresis coupled with fluorography and autoradiography. In dark-imbibed spores, RNA synthesis was initiated about 24 h after sowing, with most of the label accumulating in the high mol. wt. poly(A) -RNA fraction. There was no incorporation of the label into poly(A) +RNA until 48 h after sowing. In contrast, photo-induced spores began to synthesize all fractions of RNA within 12 h after sowing and by 24 h, incorporation of 3H-uridine into RNA of irradiated spores was nearly 70-fold higher than that into dark-imbibed spores. Protein synthesis, as monitored by 3H-arginine incorporation into the acid-insoluble fraction and by autoradiography, was initiated in spores within 1-2 h after sowing under both conditions. Autoradiographic experiments also showed that onset of protein synthesis in the cytoplasm of the germinating spore is independent of the transport of newly synthesized nuclear RNA. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of 35S-methionine-labelled proteins revealed a good correspondence between proteins synthesized in a cell-free translation system directed by poly(A) +RNA of dormant spores and those synthesized in vivo by dark-imbibed and photo-induced spores. These results indicate that stored mRNAs of O. sensibilis spores are functionally competent and provide templates for the synthesis of proteins during dark-imbibition and germination.     

Cooperative enhancement at the Drosophila Sgs-3 locus

The Drosophila glue gene Sgs-3 is specifically expressed in the secretory cells of the salivary glands of third instar larvae. We have assayed the expression of gene fusions to determine the role of cis-acting Sgs-3 sequences in conferring this pattern of expression. These experiments define two regulatory regions required for expression of reporter genes from the Sgs-3 promoter. One region, between 106 and 56 bp upstream of the Sgs-3 mRNA 5' end is sufficient for low but correct tissue- and stage-specific expression. A second region, lying between 629 and 130 bp 5' of the RNA start site is functionally equivalent; that is, it alone will also direct low level, specific expression. These two regions act synergistically to give high level expression. More distant upstream regions function to further increase levels of expression. These two regulatory elements can confer a salivary gland-specific pattern of expression on a heterologous promoter and are also sufficient to drive gene expression in other Drosophila species, implying conservation of regulators.     

THE SPORE-GERMINATION PATTERN OF THELYPTEROID FERNS

Cell division patterns in germinating spores of several Thelypteris species were studied using light microscopy of sectioned material and scanning electron microscopy. All species exhibited the same basic germination pattern, characterized by an asymmetric cell division of the spore parallel to the equatorial plane to delimit a proximal rhizoid, followed by a perpendicular division of the basal cell to form the protonemal cell. While spore-germination patterns appear to be a potentially useful taxonomic character in some groups of ferns, the homogeneity in this character exhibited by the thelypteroid group impairs its usefulness in the taxonomy of Thelypteris.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. V. effects of proteases on the ciliary membrane

The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the ‘immobilization antigen’, constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.