Hybridization and karyotype variability of three endemic Fritillaria L. (Liliaceae) in Argolis Peninsula (Greece)

Abstract

The Argolis Peninsula covers the north-eastern part of Peloponnisos and is surrounded by the Gulf of Argosaronic. The area hosts three species of the genus Fritillaria: F. graeca, F. rhodocanakis and F. spetsiotica. Fritillaria graeca is a Greek endemic taxon and its distribution includes Peloponnisos, C & E Sterea Ellas, C Evia and in proximity to Sterea Ellas, Salamis and Kea islands, while the stenoendemic F. rhodocanakis and F. spetsiotica are mainly found on Idra and Spetses islands respectively. The last two taxa are included in the Red Data Book of Rare and Threatened Plants of Greece, while F. rhodocanakis is also included in the IUCN Red List. Hybridization among them is a common phenomenon in the areas where they coexist, leading to an array of morphologically and karyologically intermediate forms. The current study presents the taxa’s karyomorphometric analysis for the first time and reveals hybrids’ cytological variety, including differences in marker chromosomes, polyploidy and the number of B-chromosomes.     

SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA.

We have reconstituted protein translocation across plasma membrane vesicles of Escherichia coli using purified proOmpA and trigger factor, a 63 kd soluble protein. Treatment of membrane vesicles with urea inactivates them for translocation unless a factor present in cytoplasmic extracts is added during the translocation reaction. Sedimentation analysis showed that the stimulatory activity is of distinctly higher mol. wt than trigger factor. Cytoplasmic extracts from a strain that greatly overproduces the SecA protein are highly enriched in the stimulatory activity for untreated membranes and restore translocation to urea-treated membranes, suggesting that this protein is the stimulatory factor. This assay was used to monitor the isolation of SecA protein from the overproducing strain. The purified protein is soluble, yet binds peripherally to membranes with high affinity and supports translocation. Using pure proOmpA, SecA protein, trigger factor and urea-treated membranes, the protein export process was resolved into binding and translocation steps. We find that proOmpA binds to membrane vesicles with or without SecA protein, but that translocation only occurs when SecA was bound prior to proOmpA.     

Binding of the 3'' terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation.

A key event in ribosomal protein synthesis is the translocation of deacylated tRNA, peptidyl tRNA and mRNA, which is catalyzed by elongation factor G (EF-G) and requires GTP. To address the molecular mechanism of the reaction we have studied the functional role of a tRNA exit site (E site) for tRNA release during translocation. We show that modifications of the 3' end of tRNAPhe, which considerably decrease the affinity of E-site binding, lower the translocation rate up to 40-fold. Furthermore, 3'-end modifications lower or abolish the stimulation by P site-bound tRNA of the GTPase activity of EF-G on the ribosome. The results suggest that a hydrogen-bonding interaction of the 3'-terminal adenine of the leaving tRNA in the E site, most likely base-pairing with 23S rRNA, is essential for the translocation reaction. Furthermore, this interaction stimulates the GTP hydrolyzing activity of EF-G on the ribosome. We propose the following molecular model of translocation: after the binding of EF-G.GTP, the P site-bound tRNA, by a movement of the 3'-terminal single-stranded ACCA tail, establishes an interaction with 23S rRNA in the adjacent E site, thereby initiating the tRNA transfer from the P site to the E site and promoting GTP hydrolysis. The co-operative interaction between the E site and the EF-G binding site, which are distantly located on the 50S ribosomal subunit, is probably mediated by a conformational change of 23S rRNA.     

Autoantibodies from patients with systemic lupus erythematosus can affect DNA and RNA synthesis in cultured cells

Sera reacting positively for anti-DNA antibodies from systemic lupus erythematosus (SLE) patients were tested for their effect on DNA and RNA synthesis in permeabilized cultured cells and isolated nuclei. The immunoglobulin fraction obtained by ammonium sulfate precipitation of serum was shown to exert considerable influence on DNA and RNA synthesis in cultured cells and nuclei. A component of this antibody population is anti-DNA. These antibodies exert different effects on DNA template activity which is a function of their conformational specificity. Intracellular penetration of autoantibodies as noted in SLE may be one of the reasons for clinical manifestations of disease in these patients.     

ProOmpA is stabilized for membrane translocation by either purified E. coli trigger factor or canine signal recognition particle

We have isolated large amounts of E. coli outer-membrane protein A precursor (proOmpA). Purified proOmpA is active in membrane assembly, and this assembly is saturable with respect to the precursor protein. A proOmpA-Sepharose matrix allows affinity isolation of trigger factor, a soluble, 63,000 dalton monomeric protein that stabilizes proOmpA in assembly competent form. Comparison of trigger factor's amino-terminal sequence with those in a computer data bank and with those encoded by sec genes, as well as groEL and heat shock gene dnaK, suggests that trigger factor is encoded by a previously undescribed gene. Trigger factor and proOmpA form a 1:1 complex that can be isolated by gel filtration. Purified canine signal recognition particle (SRP) can also stabilize proOmpA for membrane insertion. This postribosomal activity of SRP suggests a unifying theme in protein translocation mechanisms.     

Natural15N abundance as a method of estimating the contribution of biologically fixed nitrogen to N2-fixing systems: Potential for non-legumes

The¹⁵N abundance of plants usually closely reflects the¹⁵N abundance of their major immediate N source(s); plant-available soil N in the case of non-N₂-fixing plants and atmospheric N₂ in the case of N₂ fixing plants. The¹⁵N abundance values of these sources are usually sufficiently different from each other that a significant and systematic difference in the¹⁵N abundance between the two kinds of plants can be detected. This difference provides the basis for the natural¹⁵N abundance method of estimating the relative contribution of atmospheric N₂ to N₂-fixing plants growing in natural and agricultural settings. The natural¹⁵N abundance method has certain advantages over more conventional methods, particularly in natural ecosystems, since disturbance of the system is not required and the measurements may be made on samples dried in the field. This method has been tested mainly with legumes in agricultural settings. The tests have demonstrated the validity of this method of arriving at semi-quantitative estimates of biological N₂-fixation in these settings. More limited tests and applications have been made for legumes in natural ecosystems. An understanding of the limits and utility of this method in these systems is beginning to emerge. Examples of systematic measurements of differences in¹⁵N abundance between non-legume N₂-fixing systems and neighbouring non-fixing systems are more unusual. In principle, application of the method to estimate N₂-fixation by nodulated non-legumes, using the natural¹⁵N abundance method, is as feasible as estimating N₂-fixation by legumes. Most of the studies involving N₂-fixing non-legumes are with this type of system (e.g., Ceanothus, Chamabatia, Eleagnus, Alnus, Myrica, and so forth). Resuls of these studies are described. Applicability for associative N₂-fixation is an empirical question, the answer to which probably depends upon the degree to which fixed N goes predominantly to the plant rather than to the soil N pool. The natural¹⁵N abundance method is probably not well suited to assessing the contribution of N₂-fixation by free-living microorganisms in their natural habitat, particularly soil microorganisms.This work was supported in part by subcontracts under grants from the US National Science Foundation (DEB79-21971 and BSR821618)     

An expressed β-tubulin gene, TUBB, is located on the short arm of human chromosome 6 and two related sequences are dispersed on chromosomes 8 and 13

The chromosomal assignments of an expressed β-tubulin gene and two related sequences have been determined by Southern blot analysis of DNA from a panel of human X Chinese hamster somatic cell hybrids cleaved with Hind III or EcoR I. Probes containing the 3′ untranslated regions of the expressed gene M40 and of pseudogene 21β were used to localize the M40 sequence (gene symbol TUBB) to chromosome 6 region 6p21 → 6pter, the 21β pseudogene (TUBBP1) to chromosome 8 region 8q21 → 8pter and a third related sequence (TUBBP2) to chromosome 13. Asynteny of expressed genes and related processed pseudogenes has now been demonstrated for several gene families.     

tRNA binding sites of ribosomes from Escherichia coli

70S tight-couple ribosomes from Escherichia coli were studied with respect to activity and number of tRNA binding sites. The nitrocellulose filtration and puromycin assays were used both in a direct manner and in the form of a competition binding assay, the latter allowing an unambiguous determination of the fraction of ribosomes being active in tRNA binding. It was found that, in the presence of poly(U), the active ribosomes bound two molecules of N-AcPhe-tRNAPhe, one in the P and the other in the A site, at Mg2+ concentrations between 6 and 20 mM. A third binding site in addition to P and A sites was observed for deacylated tRNAPhe. At Mg2+ concentrations of 10 mM and below, the occupancy of the additional site was very low. Dissociation of tRNA from this site was found to be rather fast, as compared to both P and A sites. These results suggest that the additional site during translocation functions as an exit site, to which deacylated tRNA is transiently bound before leaving the ribosome. Since tRNA binding to this site did not require the presence of poly(U), a function of exit site bound tRNA in the fixation of the mRNA appears unlikely. Both the affinity and stability of binding to the additional site were found lower for the heterologous tRNAPhe from yeast as compared to the homologous one. This difference possibly indicates some specificity of the E. coli ribosome for tRNAs from the same organism.     

A Novel Photoaffinity Ligand for Kainate Sites Labels Two Polypeptides with Molecular Mass 45 and 33.5 kDa in Chick Cerebellum

Abstract: The synthesis of (2 S ,3 S ,4 S )-4-[1-(4-azidobenzamidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [³H]CNQX and [³H]kainic acid binding on chick cerebellar membranes. [³H]ABCPA was photoaffinity cross-linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [³H]ABCPA incorporation in both bands was completely blocked by 2 m M CNQX. When photoaffinity labeling was performed in the presence of 2 m M kainic acid, incorporation of [³H]ABCPA was blocked by ∼70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 m M glutamate.