Plastocyanin of Arabidopsis thaliana; isolation and characterization of the gene and chloroplast import of the precursor protein.

A genomic clone encoding the plastocyanin precursor was isolated from an Arabidopsis thaliana lambda EMBL3 library, with the help of a heterologous hybridization probe. The nucleotide (nt) sequence encoding the 171-amino acid precursor protein and 650 bp of the 5'-flanking region were determined. S1 nuclease mapping showed the Arabidopsis coding region to be uninterrupted and the transcript to possess an untranslated leader of approx. 30 nt. The 5' region of the gene contains a 25-bp direct repeat at a distance of 300 bp upstream from the ATG start codon. Southern analysis of several genomic digests shows the presence of a single copy of the plastocyanin gene in the Arabidopsis genome. In vitro synthesized pre-plastocyanin was used in import experiments with isolated pea chloroplasts. Plastocyanin was correctly directed to the thylakoid lumen and processed to the mature size. A clear single processing intermediate, as was found with the import of Silene pratensis pre-plastocyanin, seems to be absent.

Effect of 1,8-dihydroxy-9-anthrone (anthralin) on rat hepatic ornithine decarboxylase activity in vivo.

Intraperitoneal injection of the non-phorbol tumor promoter anthralin (1,8-dihydroxy-9-anthrone) in male rats resulted in an increase of hepatic ornithine decarboxylase (ODC) activity. Maximal activity was observed 8 h after promoter administration reaching levels about 30 times over control. The kinetics of anthralin dependent ODC induction differed markedly from that by either 12-O-tetradecanoylphorbol-13-acetate (TPA) or phenobarbital (PB) (Bisschop et al., Carcinogenesis 2 (1981) 1282). With anthralin a slow decrease of ODC back to control level is observed approximately within 22 h. In contrast, ODC induction mediated by other tumor promoters like TPA and PB decreased to control levels within 4-6 hours. Administration of a second dose of anthralin 8 h after the first dose prevented the activity decrease as normally observed after a single dose of a tumor promoter. This effect lasted at least 10 h. ODC activity induction occurred in a dose-dependent manner being linear from 10-2000 micrograms anthralin/kg body wt. Pretreatment of the animals either with actinomycin D or with cycloheximide completely blocked anthralin mediated ODC induction suggesting that de novo ODC-mRNA synthesis and subsequent translation is involved in this process.

Behavior of the H atom velocity distribution function within the shock wave of a hydrogen plasma jet.

The evolution of the ground-state hydrogen atom velocity distribution function throughout the stationary shock wave of a supersonic hydrogen plasma jet (3

Transport of ground-state hydrogen atoms in a plasma expansion.

The transport of ground-state atomic hydrogen in the expansion of a thermal plasma generated from an Ar-H2 mixture is studied by means of laser-based diagnostic techniques. The flow of hydrogen atoms is investigated by two-photon excitation laser-induced fluorescence (LIF), whereas Ar atoms are probed by LIF as well as by UV Rayleigh scattering. The transport of Ar atoms can be fully understood in terms of a free jet flow; H atoms on the contrary exhibit an anomalous behavior. In the course of the plasma expansion, hydrogen atoms decouple from the argon fluid by a diffusion process as a direct consequence of recombination of H atoms at the vessel walls. In this contribution it is shown, on the basis of experimental results, how plasma-surface interactions can strongly influence the flow pattern of an atomic radical fluid.

Structure of U2 snRNA genes of Arabidopsis thaliana and their expression in electroporated plant protoplasts.

We have characterized the U2 snRNA gene family in the higher plant Arabidopsis thaliana. It consists of 10-15 genes which do not appear to be closely clustered. Six of the U2 genes were sequenced and the structure of the Arabidopsis U2 RNA termini was determined in order to define the coding regions. Each of the genes codes for a distinct RNA differing from the others by 2-13 point mutations, localized in the 3' part of the 196 nt-long RNA. The upstream non-coding regions of all genes show strong sequence similarity in positions -81 to -1 and contain three highly conserved sequence elements: GTCCCACATCG (positions -78 to -68; 100% conservation), GTAGTATAAATA (-37 to -26) and CAANTC (-6 to -1). The coding regions are followed by the sequence CAN(7-9)AGTNNAA, a putative termination signal. The expression of three of the genes was studied in electroporated Orychophragmus violaceus and Nicotiana tabacum protoplasts. The genes, one of which contains a T --> C change in the Sm antigen binding site, were actively transcribed and processed into U2 RNAs of the expected size and containing trimethylguanosine caps. Deletion analysis indicates that sequences upstream of the conserved -80 to -1 region are not important for transcription in protoplasts. The 5'-terminal parts of U2 RNAs from several monocot and dicot plants were sequenced. This region, containing the sequence implicated in base-pairing with the branch point in pre-mRNA introns, is identical in all U2 RNAs examined.

A U-snRNA gene-specific upstream element and a -30 'TATA box' are required for transcription of the U2 snRNA gene of Arabidopsis thaliana.

The U2 and U5 snRNA genes of Arabidopsis thaliana contain in their promoter regions two elements with conserved sequence and position. To test the significance of this conservation we have made a construction in which the promoter of the U2 RNA gene is replaced by the synthetic 98 bp long sequence containing the two conserved elements: an upstream sequence element, GTCCCACATCG (USE, pos. -78 to -68), and a TATA-like sequence TATAAATA (-33 to -26), positioned approximately three helical turns apart, as in the wild-type promoter. This synthetic promoter efficiently drove transcription of the U2 gene in transfected protoplasts of Nicotiana plumbaginifolia. The importance of the individual elements and of their position within the promoter was investigated. Deletion of the USE, change of its orientation, and some single point mutations all decreased transcription 10- to 20-fold, and replacement of the TATA-like element by an unrelated sequence inactivated the promoter. Mutants in which the spacing between the USE and TATAAATA was changed were less active but no correlation was observed between promoter activity and insertion of either odd or even numbers of half helical turns. Insertion of a spacer between TATAAATA and the cap site resulted in accumulation of U2 RNA with an extended 5' end, indicating that the TATAAATA element is responsible for selection of the initiation site. The data indicate that the promoters of RNA polymerase II-specific U-snRNA genes in higher plants differ from their animal counter-parts and also from plant mRNA gene promoters. They contain two essential elements, an USE, an element found only in U-snRNA genes, and a TATA element which is indistinguishable from the TATA boxes of mRNA-coding genes.

Structure and expression of the U5 snRNA gene of Arabidopsis thaliana. Conserved upstream sequence elements in plant U-RNA genes.

We have previously characterized the U2 small nuclear (sn) RNA gene family of Arabidopsis thaliana. To find out the structural features of upstream and downstream non-coding regions that are shared by different U-RNA genes in higher plants we have isolated the gene encoding a 125 nt-long U5 snRNA of Arabidopsis. Activity of the cloned gene was demonstrated in stably transformed tobacco calli and by transient expression in transfected protoplasts of Nicotiana plumbaginifolia. Southern analysis indicated that the Arabidopsis genome contains 8-9 copies of the U5 gene. Alignment of upstream non-coding regions revealed two elements conserved between all plant U-RNA genes characterized so far: the sequence RTCCCACATCG (-70/-80 region, 100% conservation) and the TATA homology around position -30. The coding regions in all genes are followed by the sequence CAN4-9AGTN (A/T)AA which may correspond to a termination and/or processing signal.

Relaxation behavior of rovibrationally excited H2 in a rarefied expansion.

The evolution of the rotational and vibrational distributions of molecular hydrogen in a hydrogen plasma expansion is measured using laser induced fluorescence in the vacuum-UV range. The evolution of the distributions along the expansion axis shows the relaxation of the molecular hydrogen from the high temperature in the upstream region to the low ambient temperature in the downstream region. During the relaxation, the vibrational distribution, which has been recorded up to v = 6, is almost frozen in the expansion and resembles a Boltzmann distribution at T approximately 2200 K. However, the rotational distributions, which have been recorded up to J = 17 in v = 2 and up to J = 11 in v = 3, cannot be described with a single Boltzmann distribution. In the course of the expansion, the lower rotational levels (J < 5) adapt quickly to the ambient temperature ( approximately 500 K), while the distribution of the higher rotational levels (J > 7) is measured to be frozen in the expansion at a temperature between 2000 and 2500 K. A model based on rotation-translation energy transfer is used to describe the evolution of the rotational distribution of vibrational level v = 2 in the plasma expansion. The behavior of the low rotational levels (J < 5) is described satisfactory. However, the densities of the higher rotational levels decay faster than predicted.

Enzymic characterization of Bacillus subtilis GTP cyclohydrolase I. Evidence for a chemical dephosphorylation of dihydroneopterin triphosphate.

GTP cyclohydrolase I catalyses the first committing step in the biosynthesis of the pterin moiety of folic acid: conversion of GTP to dihydroneopterin triphosphate. GTP cyclohydrolase I of Bacillus subtilis was purified to homogeneity and shown to have a homo-octameric structure. The enzyme had an apparent Km for GTP of 4 microM and, in the absence of cations, a Vmax. of 80 nmol/min per mg of protein. K+ ions moderately increased its Vmax., whereas UTP and Ca2+ and Mg2+ ions drastically increased its Km for GTP. Dihydrofolate and other products of the folate and tetrahydrobiopterin pathways did not inhibit GTP cyclohydrolase I. In addition to their effect on the enzyme activity, Ca2+ and Mg2+ ions catalysed the chemical dephosphorylation of dihydroneopterin triphosphate to non-cyclic dihydroneopterin monophosphate, the substrate for the phosphomonoesterase reaction in folate biosynthesis. This dephosphorylation was specific and did not require the action of a phosphatase. We suggest a physiological role for Ca2+ ions and UTP in regulation of folate biosynthesis at the levels of GTP cyclohydrolase I and dephosphorylation of dihydroneopterin triphosphate.

Domains of U4 and U6 snRNAs required for snRNP assembly and splicing complementation in Xenopus oocytes.

Structure-function relationships in the vertebrate U4-U6 snRNP have been analysed by assaying the ability of mutant RNAs to form U4-U6 snRNPs and to function in splicing complementation in Xenopus oocytes. The mutants define three categories of domain within the RNAs. First, domains which are not essential for splicing. These include regions of U6 which have previously been implicated in the capping and transport to the nucleus of U6 RNA as well as, less surprisingly, regions of U4 and U6 which have been poorly conserved in evolution. Second, domains whose mutation reduces U4-U6 snRNP assembly or stability. This group includes mutations in both the proposed U4-U6 interaction domain, and also, in the case of U6, in a highly conserve sequence flanking stem I of the interaction domain. These mutants are all defective in splicing. Third, regions not required for U4-U6 assembly, but required for splicing complementation. This category defines domains which are likely to be required for specific contacts with other components of the splicing machinery. Combinations of mutants in the U4 and U6 interaction domain are used to show that there are not only requirements for base complementarity but also for specific sequences in these regions.

Roles of U4 and U6 snRNAs in the assembly of splicing complexes.

A series of U4 and U6 snRNA mutants was analysed in Xenopus oocytes to determine whether they block splicing complex assembly or splicing itself. All the U4 and U6 mutants found to be inactive in splicing complementation resulted in defects in assembly of either U4/U6 snRNP or of splicing complexes. No mutants were found to separate the entry of U5 and U6 snRNAs into splicing complexes and neither of these RNAs was able to associate with the pre-mRNA in the absence of U4. In the absence of U6 snRNA, however, U4 entered a complex containing pre-mRNA as well as the U1 and U2 snRNAs. U6 nucleotides whose mutation resulted in specific blockage of the second step of splicing in Saccharomyces cerevisiae are shown not to be essential for splicing in the oocyte assay. The results are discussed in terms of the roles of U4 and U6 in the assembly and catalytic steps of the splicing process.