Processing of the intron-encoded U16 and U18 snoRNAs: the conserved C and D boxes control both the processing reaction and the stability of the mature snoRNA.

A novel class of small nucleolar RNAs (snoRNAs), encoded in introns of protein coding genes and originating from processing of their precursor molecules, has recently been described. The L1 ribosomal protein (r-protein) gene of Xenopus laevis and its human homologue contain two snoRNAs, U16 and U18. It has been shown that these snoRNAs are excised from their intron precursors by endonucleolytic cleavage and that their processing is alternative to splicing. Two sequences, internal to the snoRNA coding region, have been identified as indispensable for processing the conserved boxes C and D. Competition experiments have shown that these sequences interact with diffusible factors which can bind both the pre-mRNA and the mature U16 snoRNA. Fibrillarin, which is known to associate with complexes formed on C and D boxes of other snoRNAs, is found in association with mature U16 RNA, as well as with its precursor molecules. This fact suggests that the complex formed on the pre-mRNA remains bound to U16 throughout all the processing steps. We also show that the complex formed on the C and D boxes is necessary to stabilize mature snoRNA.     

The beta-amyloid domain is essential for axonal sorting of amyloid precursor protein.

We have analysed the axonal sorting signals of amyloid precursor protein (APP). Wild-type and mutant versions of human APP were expressed in hippocampal neurons using the Semliki forest virus system. We show that wild-type APP and mutations implicated in Alzheimer's disease and another brain beta-amyloidosis are sorted to the axon. By analysis of deletion mutants we found that the membrane-inserted APP ectodomain but not the cytoplasmic tail is required for axonal sorting. Systematic deletions of the APP ectodomain identified two regions required for axonal delivery: one encoded by exons 11-15 in the carbohydrate domain, the other encoded by exons 16-17 in the juxtamembraneous beta-amyloid domain. Treatment of the cells with the N-glycosylation inhibitor tunicamycin induced missorting of wild-type APP, supporting the importance of glycosylation in axonal sorting of APP. The data revealed a hierarchy of sorting signals on APP: the beta-amyloid-dependent membrane proximal signal was the major contributor to axonal sorting, while N-glycosylation had a weaker effect. Furthermore, recessive somatodendritic signals, most likely in the cytoplasmic tail, directed the protein to the dendrites when the ectodomain was deleted. Analysis of detergent solubility of APP and another axonally delivered protein, hemagglutinin, demonstrated that only hemagglutinin formed CHAPS-insoluble complexes, suggesting distinct mechanisms of axonal sorting for these two proteins. This study is the first delineation of sorting requirements of an axonally targeted protein in polarized neurons and indicates that the beta-amyloid domain plays a major role in axonal delivery of APP.     

NADP+-isocitrate dehydrogenase in germinating cucumber cotyledons: Purification and characterization of a cytosolic isoenzyme

The activity of NADP⁺-dependent isocitrate dehydrogenase (ICDH, EC 1.1.1.42) was investigated during the post-germinative growth of cucumber ( Cucumis sativus L. cv. Marketmore) seedlings. Isoelectric focusing showed the presence of several isoenzymes, two of which represented 70–80% of the total NADP⁺-ICDH activity in cotyledons of seedlings grown in the dark. They had pI values between 4.8 and 5.8. The isoenzyme with higher pI was purified to homogeneity by hydrophobic interaction, affinity, hydroxylapatite and anion exchange chromatography. The purified isoenzyme is a dimeric protein, consisting of two apparently identical 43-kDa subunits. It is specific for NADP⁺, inhibited by ATP and by 2-oxoglutarate, whereas it is not inhibited by citrate, succinate, and glyoxylate. The data indicate that NADP⁺-ICDH from cucumber is structurally similar to ICDHs from other plants, but it shows some peculiar biochemical characteristics.     

Dynamics of energy and nutrient concentration and construction cost in a native and two alien C4 grasses from two neotropical savannas

In Venezuela, the alien grasses Melinis minutiflora Beauv. and Hyparrhenia rufa (Nees.) Stapf tend to displace the native savanna plant community dominated by Trachypogon plumosus (Humb. and Bonpl.) Nees. This occurs in either relatively wetter and fertile highland savannas or in drier and less fertile lowland savannas. Although the native and aliens are perennial C₄ grasses, higher net assimilation leaf biomass per plant and germination rate of the latter are some causes for their higher growth rates and for their competitive success. The objective of this study is to compare seasonal tissue energy, N, P and K concentrations and the calculated construction costs (CC) between the native grass and either one of the alien grasses from lowland and highland savannas. We predict that, in order to out-compete native plants, alien grasses should be more efficient in resource use as evidenced by lower tissue energy and nutrient concentrations and CC.Tissue energy and nutrient concentration were measured throughout the year and compared between M. minutiflora and the co-occurring local population of T. plumosus in a highland savanna and between H. rufa and its neighbor local population of T. plumosus in a lowland savanna. CC was calculated from energy, N and ash concentrations considering ammonium as the sole N source. Differences between co-occurring species, T. plumosus populations, seasons, and organs were analyzed with ANOVA.Highland and lowland grasses differed in concentration and allocation of energy and nutrients whereas the differences between alien and native grasses were specific for each pair considered. Highland grasses had higher energy, N, P and CC than lowland grasses. These variables were always lowest in the culms. In the more stressed lowland site, tissue energy and nutrient concentrations decreased significantly during the dry season except in the roots of both grasses which had the highest energy and nutrients concentrations during the drought. This seasonal response was more marked in the local lowland population of T. plumosus in which maximum CC alternated seasonally between leaves and roots. Energy and nutrient concentrations and CC were the lowest in H. rufa. In the lowland savannas, the higher efficiency of resource use in the invader grass contributes to its higher competitive success through increased growth rate. In the highlands, overall tissue energy concentration and CC, but not N nor P concentration, were lower in the fast growing M. minutiflora but seasonal differences were lacking. The higher leaf CC in T. plumosus can be attributed to the higher proportion of sclerenchyma tissue which is more expensive to construct. Considering CC, both fast growing alien grasses are more efficient in resource use than the co-occurring native grass. However, the role of CC explaining the competitive success of the former, through higher growth rates, is more evident in the more stressful environment of the lowland savanna.     

Frequencies,Timing, and Spatial Patterns of Co-Suppression of Nitrate Reductase and Nitrite Reductase in Transgenic Tobacco Plants

Frequencies, timing, and spatial patterns of co-suppression of the nitrate (Nia) and nitrite (Nii) genes were analyzed in transgenic tobacco (Nicotiana tabacum) plants carrying either Nia or Nii cDNAs under the control of the 35S promoter, or a Nii gene with its own regulatory signals (promoter, introns, and terminator) cloned downstream of two copies of the enhancer of the 35S promoter. We show that (a) the frequencies of transgenic lines affected by co- suppression are similar for the three constructs, ranging from 19 to 25%; (b) Nia and Nii co-suppression are triggered stochastically during a phenocritical period of 2 weeks between germination and flowering; (c) the timing of co-suppression (i.e. the percentage of isogenic plants affected by co-suppression reported as a function of the number of days of culture) differs from one transgenic line to another; (d) the percentage of isogenic plants affected by co-suppression is increased by growing the plants in vitro prior to their transfer to the greenhouse and to the field; and (e) at the end of the culture period, plants are either unaffected, completely co-suppressed, or variegated. Suppressed and nonsuppressed parts of these variegated plants are separated by a vertical plane through the stem in Nia co-suppression, and separated by a horizontal plane in Nii co-suppression.     

Selection of monosomic addition plants in offspring families using repetitive DNA probes in Beta L.

The distribution of two repetitive DNA probes Sat-121 and PB6-4, specific for the section Procumbentes of the genus Beta, was tested in 16 B. patellaris monosomic addition families using a dot-blot hybridization procedure. All monosomic additions were accurately distinguished from diploid sib plants with both DNA probes. The probe PB6-4, with the strongest signal after hybridization, was selected for rapid screening of an extensive number of putative monosomic additions in B. patellaris or B. procumbens addition families using a squash-blot hybridization procedure. The probe PB6-4 detected 118 monosomic additions in 640 plants (18.4%) in eight different B. procumbens addition families. The addition family with chromosome 4 of B. procumbens was semi-lethal and could not be tested. The distribution of PB6-4 in B. patellaris addition families was confirmed in 63 addition families using the squash-blot procedure. In 4580 plants of these addition families, 628 individual monosomic additions (13.7%) were found. The relationship of the morphological characteristics of monosomic addition plants to the results of the squash-blot hybridization (plants with signal) using probe PB6-4 is quite rigorous but not complete. The correlation between plants with a signal and chromosome number (2n=19) is complete. These results indicate that sequences present on PB6-4 are probably present on all chromosomes of B. patellaris and B. procumbens. The possibility of utilizing the sequence information of Sat-121 for a PCR-based assay to screen for putative monosomic addition plants was also investigated as an alternative to chromosome counting. The DNA-amplification profiles using the primers REP and REP.INV clearly distinguished monosomic addition plants from their diploid sibs.     

Kinetics of an autocatalytic zymogen reaction in the presence of an inhibitor coupled to a monitoring reaction

A global kinetic analysis of a model consisting of an autocatalytic zymogen-activation process, in which an irreversible inhibitor competes with the zymogen for the active site of the proteinase, and a monitoring coupled reaction, in which the enzyme acts upon one of its substrates, is presented. This analysis is based on the progress curves of any of the two products released in the monitoring reaction. The general solution is applied to an important particular case in which rapid equilibrium conditions prevail. Finally, we suggest a procedure to predict whether the inhibition or activation route dominates in the steady state of the system. These results generalize our previous analysis of simpler mechanisms.     

Complete sequence of a new HLA-B35 allele found in a tribe of Mapuche Indians in the south of Argentina

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U17107. The nameB*3509 was officially assigned by the WHO Nomenclature Committee in December 1994     

14-3-3 Protein homologues play a central role in the fusicoccin signal transduction pathway

The plasma membrane located fusicoccin binding protein (FCBP) is an essential element in the fusicoccin (FC) signal transduction pathway. We obtained primary sequence information for the 31 kD subunit of the FCBP. These sequences showed that the FCBP is homologous to members of the 14-3-3 protein family. Both the 31 and 30 kD subunits cross-react with 14-3-3 antibodies. In native form the FCBP occurs as a dimer, but it is also part of a complex with higher molecular mass. The monomeric forms of the FCBP (the 30 and 31 kD subunits) do not have ³H-FC binding activity. We discuss how the FCBP, as a member of the 14-3-3 protein family, may be able to bind FC and how the FC-signal is transduced to the effector protein, the H⁺-ATPase.     

Galanthamine production in “shoot-clump” cultures of Narcissus confusus in liquid-shake medium

Galanthamine (GAL) is increasingly used in the treatment of Alzheimer's disease. We have attempted to develop a method of producing this alkaloid using in vitro cultures of Narcissus confusus plants. The “shoot-clump” culture in liquid medium was shown to be an appropriate method for the micropropagation of this bulbous plant. The complete process included three steps:

1. culture of “twin-scales” starting from the bulbs;
2. culture of the newly formed shoots in a medium for bud proliferation (Murashige Skoog+1 mg l^-1 of 2,4-dichlorophenoxyacetic acid+5 mg l^-1 of benzyladenine), and
3. culture of “shoot-clumps” in a liquid-shake medium. Here we describe the effect of the addition of trans-cinnamic acid, a precursor in the biosynthesis of the Amaryllidaceae alkaloids, on the production of galanthamine and related alkaloids, and also on the growth of the “shoot-clump” culture. The production of galanthamine was found to be inhibited by the addition of the precursor, which promoted the production of the other alkaloid in the same biosynthetic pathway, N-formyl-norgalanthamine. The total production of galanthamine in the control cultures in day-long photoperiod was 2.50 mg per culture, of which 1.97 mg per culture were released into the medium.