[Characteristics of the signal lag effect on crew--control center communications in the 520-day simulation experiment].

The 520-day experimental simulation of an exploration mission provided an opportunity to apply content analysis for studying the patterns of crew--Control center (CC) communication impeded by lag times. The period of high autonomy was featured by drastic reduction of the number of crew questions and requests which was judged as a marker of adaptation to the simulated space mission environment. The "key" events in the experiment changed the content of crew messages radically attesting to misperception of time, emotional involvement, want of CC feedback and draining out negative emotions. After the period of high autonomy with full loss of communication with controllers the traffic of crew messages onto the outside was noted to become very light which could also point to temporal changes in the communication style developed in the conditions of isolation and autonomous existence.

Characterization of protein and transcript levels of the chaperonin containing tailless complex protein-1 and tubulin during light-regulated growth of oat seedlings.

In grass seedlings the network of cortical microtubules is reorganized during light-dependent growth of coleoptiles and mesocotyls. We investigated the effects of light-dependent growth on the relative steady-state levels of the mRNAs and protein levels of alpha-tubulin and the epsilon-subunit of the chaperonin containing tailless complex protein-1 in oat (Avena sativa) coleoptiles, which were grown in different light conditions to establish different growth responses. The soluble pools of the epsilon-subunit of the chaperonin containing tailless complex protein-1 and alpha-tubulin decreased in nonelongating coleoptiles, suggesting that the dynamics of the light-regulated soluble pool reflect the processes occurring during reorganization of cortical microtubules. The shifts in pool sizes are discussed in relation to the machinery that controls the dynamic structure of cortical microtubules in plant cells.

The third member of the Tetrahymena CCT subunit gene family, TpCCT alpha, encodes a component of the hetero-oligomeric chaperonin complex.

The sequence of a third member of the Tetrahymena pyriformis chaperonin CCT ('chaperonin containing TCP1') subunit gene family is presented. This gene, designated TpCCT alpha, is the orthologue of the mouse chaperonin gene TCP1/CCT alpha. To characterize the CCT complex in this ciliate, we have produced polyclonal antibodies against synthetic peptides based on C-terminal sequences deduced from the primary sequences of the TpCCT alpha, TpCCT gamma and TpCCT eta subunits. We have also used polyclonal antibodies produced against recombinant yeast CCT alpha and CCT beta subunits. Using these antibodies, we show that Tetrahymena cells contain a hetero-oligomeric CCT chaperonin comprising at least seven distinct subunits. Three of these were assigned to specific TpCCT genes, whereas a fourth was recognized by the polyclonal antibody against yeast CCT beta, suggesting that this gene is also present in the ciliate. The CCT complex also contains other unidentified proteins that were recognized by the polyclonal antibody UM-1, raised against the putative ATP binding domain of the chaperonin proteins. TpCCT alpha gene expression was shown in exponentially growing cells and cells regenerating their cilia for different periods to have a similar pattern to the previously identified genes TpCCT gamma and TpCCT eta, and also to tubulin genes.

Developmental and light-dependent changes of the cytosolic chaperonin containing TCP-1 (CCT) subunits in maize seedlings, and the localization in coleoptiles.

The cytosolic chaperonin containing TCP-1 (CCT) is known to keep fold cytoskeletal proteins and is involved in the proper organization of the cytoskeleton. These studies are based on the assumption that growth responses linked to structural rearrangement of the plant cytoskeleton include the action of CCT and the need for newly synthesized tubulin. The presence of the alpha- and epsilon-subunits of CCT was investigated in soluble fractions of protein extracts from maize mesocotyls and coleoptiles at distinct growth stages. The CCT-subunits, tubulins and actin decreased in the coleoptile in response to far-red light. In addition, independent from light treatment, the amount of CCT epsilon abundance declined with age in coleoptiles and mesocotyls between 2 and 4.5 days after sowing. In contrast to CCT epsilon, no significant light regulation of CCT alpha was found in the mesocotyl. In two day old, light-grown rapidly elongating coleoptiles part of the CCT alpha subunit and the bulk of actin and tubulin was found shifted into fractions of high molecular weight complexes when compared to slowly elongating, dark grown coleoptiles. In 4.5 day old, etiolated and elongating coleoptiles, part of both CCT-subunits and cytoskeleton proteins were found in fractions of high molecular weight. A complete disappearance of these polypeptides was observed in old far-red irradiated growth-arrested coleoptiles. CCT epsilon was found to be colocalized to microtubular structures and to the nucleus. We conclude from our data that abundance of CCT-subunits in soluble extracts is dependent on age and light treatment, but independent from the growth stage of mesocotyl and coleoptile.

A human homolog of the mitochondrial protein import receptor Mom19 can assemble with the yeast mitochondrial receptor complex.

Import of preproteins into mitochondria requires transport machineries in both mitochondrial membranes that have been characterized in Saccharomyces cerevisiae and Neurospora crassa. By cDNA analysis, we identified a human protein of 16 kDa with significant overall homology to the fungal mitochondrial import receptor Mom19, including the three typical characteristics: a hydrophobic N-terminal segment, a tetratrico peptide motif in the middle and a negatively charged C-terminus. The human Mom19 homolog is expressed in all tissues analyzed. When synthesized in vitro, the human Mom19 homolog is targeted to isolated yeast mitochondria and specifically associates with the outer membrane receptor complex, suggesting that indeed a mitochondrial import receptor was identified.

Ultraviolet Light Inhibition of Phytochrome-Induced Flavonoid Biosynthesis and DNA Photolyase Formation in Mustard Cotyledons (Sinapis alba L.).

In cotyledons of etiolated mustard (Sinapis alba L.) seedlings, phytochrome-far-red-absorbing form-induced flavonoid biosynthesis was found to be inhibited by short-term ultraviolet (UV) irradiations. UV inhibition was shown for the synthesis of quercetin, anthocyanin, and also for the accumulation of the mRNA for chalcone synthase, the key enzyme of this pathway. The UV effect was more pronounced on flavonoid biosynthesis, a process that selectively occurs in the epidermal layers, than on the synthesis of mRNA for chlorophyll a/b-binding protein localized in the mesophyll tissue. These UV inhibitory effects were accompanied by cyclobutane pyrimidine dimer (CPD) formation showing a linear fluence-response relationship. CPD formation and UV inhibition of flavonoid biosynthesis was found to be partially reversible by blue/UV-A light via DNA photolyase (PRE), allowing photoreactivation of the DNA by splitting of CPDs, which are the cause of the UV effect. Like flavonoid formation PRE was also induced by the far-red-absorbing form of phytochrome and induction was inhibited by UV. A potential risk of inhibition, in response to solar UV-B irradiation, was shown for anthocyanin formation. This inhibition, however, occurred only if photoreactivation was experimentally reduced. The PRE activity present in the etiolated seedlings (further increasing about 5-fold during light acclimatization) appears to be sufficient to prevent the persistence of CPDs even under conditions of high solar irradiation.

Regulation of phytochrome A mRNA abundance in parsley seedlings and cell-suspension cultures.

A cDNA clone encoding the apoprotein of a parsley phytochrome was isolated and classified as parsley PHYA phytochrome, on the basis of a sequence homology comparison with all available phytochrome sequences. Red light pulses led to a phytochrome-dependent down-regulation of PHYA mRNA abundance in etiolated parsley seedlings to a level of 10-20% compared with the dark control. The PHYA mRNA abundance in a parsley cell suspension culture was also down-regulated by light pulses. Transient expression assays in parsley protoplasts showed light regulation of a chimeric pea PHYA promoter uidA-gene construct.

Deletion of the receptor MOM19 strongly impairs import of cleavable preproteins into Saccharomyces cerevisiae mitochondria.

The mitochondrial outer membrane proteins MOM19 and MOM72 are thought to function as import receptors for nuclear encoded preproteins. Different views exist about the importance of each receptor in the import of cleavable and noncleavable preproteins into mitochondria. Here we cloned and sequenced MOM19 from Saccharomyces cerevisiae and constructed a gene disruption mutant. Yeast cells lacking MOM19 were unable to grow on nonfermentable carbon sources and were slow in growing on a fermentable medium, while the growth of yeast cells lacking MOM72 (Mas70p) was much less impaired. delta MOM19 cells accumulated considerable amounts of mitochondrial preproteins in vivo. The import of cleavable preproteins into isolated delta MOM19 mitochondria was strongly inhibited, while import of the noncleavable ADP/ATP carrier and phosphate carrier was only slightly inhibited. The reciprocal situation was found for protein import into delta MOM72 mitochondria. In particular, import of the cleavable precursor of cytochrome c1 into delta MOM72 mitochondria was, in agreement with a previous report (Hines, V., and Schatz, G. (1993) J. Biol. Chem. 268, 449-454), found to be partially inhibited, yet a much stronger inhibition of import was seen into delta MOM19 mitochondria. The direct comparison of protein import into yeast mutants of either receptor yields a unifying hypothesis on mitochondrial preprotein targeting; both receptors have an overlapping specificity, and MOM19 plays a major role for cleavable preproteins. Interestingly, the primary sequence of MOM19 predicts the presence of a tetratricopeptide motif that was also found in MOM72, in the peroxisomal membrane protein PAS8/PAS10, and in several proteins involved in RNA synthesis or mitosis.

Two Tcp-1-related but highly divergent gene families exist in oat encoding proteins of assumed chaperone function.

Tcp-1-related sequences have been isolated from a cDNA library of etiolated 6-day-old oat (Avena sativa) seedlings. This attempt was made to obtain cDNAs of a recently published 60 kDa plant chaperone that re-folds denatured phytochrome and which was biochemically characterised as a Tcp-1-related protein [(1993) Nature 363, 644-647]. The translation of the putative coding sequence from one full-length cDNA clone displays no specific homologies to amino acid sequences known from peptide sequencing of the oat 60 kDa chaperone. Antibodies raised against the 60 kDa chaperone and over-expressed protein from one full-length coding sequence for Tcp-1 from oat show no cross-reactivity, whereas a monoclonal antibody raised against mouse Tcp-1 protein recognizes both the 60 kDa protein purified from plant extracts and over-expressed protein from Tcp-1-related cDNA sequences.

Wound-induced systemic accumulation of a transcript coding for a Bowman-Birk trypsin inhibitor-related protein in maize (Zea mays L.) seedlings.

The wound-induced accumulation of a transcript coding for a polypeptide of 72 amino acid residues in maize (Zea mays L.) seedlings was investigated. Sequence comparison showed strong homology with Bowman-Birk trypsin inhibitors of cereal and legume species. The local accumulation pattern of this transcript demonstrated, after wounding various parts of the seedling, a local as well as a systemic response which implies the transmission of a wound signal specifically from the lower to the upper parts of the plant.

Cell Communication, Stochastic Cell Responses, and Anthocyanin Pattern in Mustard Cotyledons.

The role of intercellular signals in plant development was investigated using phytochrome-induced formation of anthocyanin in cotyledons of white mustard as a model system. The problem was approached by irradiating different subregions of the cotyledon with a microbeam. This technique was combined with in situ hybridization of chalcone synthase mRNA after irradiation of the entire cotyledon. Individual cells that exhibited all-or-none responses with a resultant stochastic, patchy pattern were examined during early stages of anthocyanin synthesis. It was demonstrated that the responses of individual cells were subsequently integrated by long-range inhibitory signals. This process led to ordered and gradually developing patterns that could be detected when final stages were analyzed at the whole-organ level. The significance of these findings is discussed in terms of efforts toward a general understanding of photomorphogenesis in plants.

Cloning and characterization of a chalcone synthase gene from mustard and its light-dependent expression.

Genomic DNA from mustard was cloned in EMBL4 and screened for chalcone synthase (CHS) genes using a heterologous cDNA probe from parsley. Two clones which hybridized with the parsley cDNA probe were isolated. They showed different restriction patterns. One clone was sequenced and identified as a CHS gene by sequence comparison with published CHS sequences. The sequence of the coding region is 1188 bp, and encodes a protein of 43 kDa. The start-point of transcription was determined by primer extension. The sequence of 0.9 kbp at the 5' end of the transcription start and part of the noncoding 3' of this gene were also determined. The coding sequence is interrupted by a single intron of 523 bp. The coding and the noncoding 5' sequence of this gene was compared with CHS genes from other species. A very high homology was found with the Arabidopsis CHS coding region. A sequence motif (CACGTGT) which is present in most rbcS and all CHS upstream regions, and which specifically binds a protein factor from plant nuclear extracts, is also present in the upstream region of the mustard CHS gene. Measurements of CHS transcript levels show that phytochrome controls expression of this gene in cotyledons of mustard seedlings; however, blue/UV-light photoreceptors control expression in later stages of development.

Development- and light-dependent regulation of the expression of two different chalcone synthase transcripts in mustard cotyledons.

Two different chalcone synthase (CHS) transcripts show similar expression characteristics under different light regimes in cotyledons of mustard (Sinapis alba L.). Etiolated seedlings show an increase in dark-expression 36-42 h after sowing. Under continuous red or far-red irradiation both CHS transcripts start to accumulate to levels above those of the dark control at 24-27 h after sowing. This time point can therefore be considered as the starting (or competence) point for phytochrome control of CHS. Continuous far-red irradiation stimulates transcript accumulation more than red light, indicating the involvement of a high-irradiance response (HIR). Irradiation of etiolated seedlings with 5 min long-wavelength far-red light (RG9) at 6-21 h after sowing decreases CHS-mRNA levels below those of the dark control. It is concluded that CHS dark-expression in etiolated seedlings is controlled by a pool of stabletype phytochrome which is derived from seed tissue. By contrast, an RG9-light pulse given to etiolated seedlings 30 h after sowing causes accumulation of CHS-mRNA above the dark-control level. This response and the HIR are attributed to the action of labile phytochrome for which the seedling becomes competent at the starting point 24-27 h after sowing. The different starting points for CHS-mRNA expression in darkness and in light (36 h and 24 h, respectively, after sowing) also indicate that the tested CHS genes in mustard are under the photocontrol of two distinct phytochrome pools. Northern analysis shows that both CHS-mRNAs are expressed in primary leaves, epicotyls and young flower buds. In-situ hybridization with gene-specific CHS probes reveals similar expression patterns for both transcripts in cotyledons of seedlings grown under 42 h continuous far-red light.

The 23 KDa polypeptide of the photosynthetic oxygen-evolving complex from mustard seedlings (Sinapis alba L.). Nucleotide sequence of cDNA and evidence for phytochrome control of its mRNA abundance.

The nucleotide sequence of a cDNA from mustard shows 78% homology in deduced amino acid sequence for the mature protein compared to the sequence for the 23 kDa protein of the oxygen-evolving complex from spinach [(1987) FEBS Lett. 216, 234-240]. There is also a high degree of homology between the premature protein sequences concerning the hydrophobic domain and its distance from the suggested processing site. The accumulation of mRNA for the 23 kDa protein in mustard was stimulated by continuous far-red light and reversal experiments by means of red/far-red light pulse treatment show the involvement of phytochrome in controlling the mRNA abundance for the 23 kDa polypeptide in mustard. The accumulation of the mRNA can be inhibited in white light if the seedlings are treated with the herbicide Norflurazon.