The cell-cycle progression of germinating embryos of maize ( Z e a m a y s L.) was studied from 0 to 72 h after the start of imbibition using DNA flow cytometry on isolated nuclei, and analyses of thymidine kinase activity, histone biosynthesis and levels of proliferating cell nulcear antigen (PCNA). At the start of germination, 75% of the cells were in G1, but this population had decreased to 25% by 72 h. The concomitant increase of cells in S-phase did not occur continuously, but stepwise, indicating that during germination most of the cells enter S-phase as a partially synchronized population. Within the initial 60 h of embryo germination the cells passed through one S-phase; the start and duration of this period of replicative DNA synthesis was further substantiated by the analysis of S-phase-associated events, the biosynthesis of core histones and the enzyme activity of thymidine kinase, which both began to increase at about 12 h after the start of differentiation. Thymidine kinase fluctuated periodically during germination with a transient maximum at 30 h and a second peak at 72 h; histone biosynthesis was not detectable until 12 h after the start of germination. The levels of PCNA protein closely resembled the pattern of thymidine kinase during germination. Together with the cytometric data this allows a clear assignment of cell cycle events to different times of embryo differentiation.

Physarum polycephalum expresses two closely related, calcium-independent NOSs (nitric oxide synthases). In our previous work, we showed that both NOSs are induced during starvation and apparently play a functional role in sporulation. In the present study, we characterized the genomic structures of both Physarum NOSs, expressed both enzymes recombinantly in bacteria and characterized their biochemical properties. Whereas the overall genomic organization of Physarum NOS genes is comparable with various animal NOSs, none of the exon–intron boundaries are conserved. Recombinant expression of clones with various N-termini identified N-terminal amino acids essential for enzyme activity, but not required for haem binding or dimerization, and suggests the usage of non-AUG start codons for Physarum NOSs. Biochemical characterization of the two Physarum isoenzymes revealed different affinities for L-arginine, FMN and 6R-5,6,7,8-tetrahydro-L-biopterin.

Physarum polycephalum is one of few non-animal organisms capable of synthesizing tetrahydrobiopterin from GTP. Here we demonstrate developmentally regulated expression of quinoid dihydropteridine reductase (EC 1.6.99.7), an enzyme required for recycling 6,7-[8H]-dihydrobiopterin. Physarum also expresses phenylalanine-4-hydroxylase activity, an enzyme that depends on dihydropteridine reductase. The 24.4 kDa Physarum dihydropteridine reductase shares 43% amino acid identity with the human protein. A number of residues important for function of the mammalian enzyme are also conserved in the Physarum sequence. In comparison to sheep liver dihydropteridine reductase, purified recombinant Physarum dihydropteridine reductase prefers pterin substrates with a 6-(1', 2'-dihydroxypropyl) group. Our results demonstrate that Physarum synthesizes, utilizes and metabolizes tetrahydrobiopterin in a way hitherto thought to be restricted to the animal kingdom.

Physarum polycephalum is an acellular slime mold. Naturally, Physarum is found in woods and meadows where it exists as macroplasmodia moving on solid surfaces and feeding from microorganisms like bacteria and yeast. Under optimal nutritional conditions plasmodia show a negative phototactical behavior. In case of food limitation, plasmodia move towards light and differentiate into sporangia finally releasing haploid spores. These spores, in proper environment, can germinate into myxamoeba, which can fuse with genetically compatible counterparts to form a zygote. Zygotes grow to macroplasmodia which are single giant cells with multiple well syncronised nuclei, therefore being of interest for cell cycle related studies. Under laboratory conditions the organism can be maintained as macroplasmodia on surfaces, or as microplasmodia in liquid shake culture. In Physarum polycephalum biosynthetic activities for the NOS cofactor tetrahydrobiopterin (BH4) as well as for folic acid precursors were found (1).

Physarum polycephalum, an acellular slime mould, serves as a model system to study cell-cycle-dependent events since nuclear division is naturally synchronous. This organism was shown to release isoxanthopterin which is structurally related to tetrahydrobiopterin, a cofactor of aromatic amino acid hydroxylases and of nitric oxide synthases (NOSs) (EC 1.14.13.39). Here, we studied Physarum pteridine biosynthesis in more detail and found that high amounts of tetrahydrobiopterin are produced and NOS activity is expressed. Physarum pteridine biosynthesis is peculiar in as much as 7,8-dihydroneopterin aldolase (EC 4.1.2.25), an enzyme of folic acid biosynthesis usually not found in organisms producing tetrahydrobiopterin, is detected in parallel. NOS purified from Physarum depends on NADPH, tetrahydrobiopterin and flavins. Enzyme activity is independent of exogenous Ca2+ and is inhibited by arginine analogues. The purified enzyme (with a molecular mass of 130 kDa) contains tightly bound tetrahydrobiopterin and flavins. During the synchronous cell cycle of Physarum, pteridine biosynthesis increases during S-phase whereas NOS activity peaks during mitosis, drops at telophase and peaks again during early S-phase. Our results characterize Physarum pteridine biosynthesis and NOS and suggest a possible link between NOS activity and mitosis.

GTP cyclohydrolase I (EC 3.5.4.16) is the first enzyme in the biosynthesis of tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H4-biopterin] in mammals and of folic acid in bacteria. Here we have characterized the GTP cyclohydrolase I gene structure and two mRNA species from Physarum polycephalum, an acellular slime mould that synthesizes H4-biopterin and metabolites of the folic acid biosynthetic pathway. Its GTP cyclohydrolase I gene consists of seven exons, and the two GTP cyclohydrolase I cDNA species isolated from Physarum encode for proteins with 228 (25.7kDa) and 195 (22.1kDa) amino acids. Furthermore, we identified two previously undescribed mRNA species in interferon-γ-treated human myelomonocytoma cells (THP-1) in addition to the cDNA coding for the fully functional 250-residue (27.9kDa) protein, which is identical with that in human phaeochromocytoma cells. One of the new splice variants codes for a 233-residue (25.7kDa) protein, whereas the other codes for the full-length protein but is alternatively spliced within the 3′-untranslated region. In heterologous expression, the shorter proteins of Physarum as well as of THP-1 cells identified here are degraded by proteolysis. Accordingly, only the 27.9kDa protein was detectable in Western blots from THP-1 cell extracts. Quantification of GTP cyclohydrolase I mRNA species in different human cell types with and without cytokine treatment showed that in addition to the correct mRNA the two splice variants isolated here, as well as the two splice variants known from human liver, are strongly induced by cytokines in cell types with inducible GTP cyclohydrolase I (THP-1, dermal fibroblasts), but not in cell types with constitutive GTP cyclohydrolase I expression (SK-N-SH, Hep-G2). As in human liver, splicing of the new mRNA variant found in THP-1 cells occurs at the boundary of exons 5 and 6. Strikingly, the 195-residue protein from Physarum is alternatively spliced at a homologous position, i.e. at the boundary of exons 6 and 7. Thus alternative splicing of GTP cyclohydrolase I at this position occurs in two species highly distant from each other in terms of evolution. It remains to be seen whether variant proteins encoded by alternatively spliced GTP cyclohydrolase I mRNA transcripts do occur in vivo and whether they participate in regulation of enzyme activity.

Drosophila epithelia acquire a planar cell polarity (PCP) orthogonal to their apical-basal axes. Frizzled (Fz) is the receptor for the PCP signal, and Dishevelled (Dsh) transduces the signal. Here, I demonstrate that unipolar relocalization of Dsh to the membrane is required to mediate PCP, but not Wingless (Wg) signaling. Dsh membrane localization reflects the activation of Fz/PCP signaling, revealing that the initially symmetric signal evolves to one that displays unipolar asymmetry, specifying the cells' ultimate polarity. This transition from symmetric to asymmetric Dsh localization requires Dsh function, and reflects an amplification process that generates a steep intracellular activity gradient necessary to determine PCP.

The myxomycetePhysarum polycephalumexpresses a calcium-independent nitric oxide (NO) synthase (NOS) resembling the inducible NOS isoenzyme in mammals. We have now cloned and sequenced this, the first nonanimal NOS to be identified, showing that it shares < 39% amino acid identity with known NOSs but contains conserved binding motifs for all NOS cofactors. It lacks the sequence insert responsible for calcium dependence in the calcium-dependent NOS isoenzymes. NOS expression was strongly up-regulated inPhysarummacroplasmodia during the 5-day starvation period needed to induce sporulation competence. Induction of both NOS and sporulation competence were inhibited by glucose, a growth signal and known repressor of sporulation, and byl-N6–(1-iminoethyl)-lysine (NIL), an inhibitor of inducible NOS. Sporulation, which is triggered after the starvation period by light exposure, was also prevented by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of NO-sensitive guanylate cyclase. In addition, also expression oflig1, a sporulation-specific gene, was strongly attenuated by NIL or ODQ. 8-Bromo-cGMP, added 2 h before the light exposure, restored the capacity of NIL-treated macroplasmodia to expresslig1and to sporulate. This indicates that the second messenger used for NO signaling in sporulation ofPhysarumis cGMP and links this signaling pathway to expression oflig1.

The mechanisms for the control of ribosomal protein synthesis have been characterized in detail in Eukarya and in Bacteria. In Archaea, only the regulation of the MvaL1 operon (encoding ribosomal proteins MvaL1, MvaL10, and MvaL12) of the mesophilic Methanococcus vannielii has been extensively investigated. As in Bacteria, regulation takes place at the level of translation. The regulator protein MvaL1 binds preferentially to its binding site on the 23S rRNA, and, when in excess, binds to the regulatory target site on its mRNA and thus inhibits translation of all three cistrons of the operon. The regulatory binding site on the mRNA, a structural mimic of the respective binding site on the 23S rRNA, is located within the structural gene about 30 nucleotides downstream of the ATG start codon. MvaL1 blocks a step before or at the formation of the first peptide bond of MvaL1. Here we demonstrate that a similar regulatory mechanism exists in the thermophilic M. thermolithotrophicus and M. jannaschii. The L1 gene is cotranscribed together with the L10 and L11 gene, in all genera of the Euryarchaeota branch of the Archaea studied so far. A potential regulatory L1 binding site located within the structural gene, as in Methanococcus, was found in Methanobacterium thermoautotrophicum and in Pyrococcus horikoshii. In contrast, in Archaeoglobus fulgidus a typical L1 binding site is located in the untranslated leader of the L1 gene as described for the halophilic Archaea. In Sulfolobus, a member of the Crenarchaeota, the L1 gene is part of a long transcript (encoding SecE, NusG, L11, L1, L10, L12). A previously suggested regulatory L1 target site located within the L11 structural gene could not be confirmed as an L1 binding site.

In Bacteria and Archaea (formerly Archaebacteria) ribosomal protein L1 has a dual function, as a primary rRNA-binding protein and as a translational repressor which binds to its own mRNA. The L1-binding site on the mRNA exhibits high similarity in both sequence and secondary structure to the binding site for L1 on the 23 S rRNA. A sensitive membrane-filter-binding assay has been used to examine the interactions between ribosomal L1 proteins from different archaeal and bacterial species, and 23S rRNA and mRNA fragments from Methanococcus vannielii containing the MvaL1-binding site. Under standard conditions (0 degrees C, pH 7.5, 20 mM Mg2+, 500 mM KCl), the apparent dissociation constant Kd of the homologous MvaL1-23S rRNA complex is 5 nM, the apparent dissociation constant Kd of the MvaL1-mRNA complex is 0.15 degrees M. L1 proteins from Escherichia coli (EcoL1) and from the thermophilic Bacterium Thermus thermophilus (TthL1), and from the thermophilic Archaea Methanococcus thermolithotrophicus (MthL1), Methanococcus jannaschii (MjaL1), and Sulfolobus solfataricus (SsoL1) were tested for their affinity to the specific L1-binding sites on the 23 S rRNA and mRNA. In general, the affinity of L1 proteins from thermophilic species to the binding sites on both 23 S rRNA and mRNA is about one order of magnitude higher than that of their mesophilic counterparts. This stronger protein-RNA interaction might make a substantial contribution to the thermal tolerance of ribosomes in thermophilic organisms.

The control of ribosomal protein synthesis has been investigated extensively in Eukarya and Bacteria. In Archaea, only the regulation of the MvaL1 operon (encoding ribosomal proteins MvaL1, MvaL10 and MvaL12) of Methanococcus vannielii has been studied in some detail. As in Escherichia coli , regulation takes place at the level of translation. MvaL1, the homologue of the regulatory protein L1 encoded by the L11 operon of E . coli , was shown to be an autoregulator of the MvaL1 operon. The regulatory MvaL1 binding site on the mRNA is located about 30 nucleotides downstream of the ATG start codon, a sequence that is not in direct contact with the initiating ribosome. Here, we demonstrate that autoregulation of MvaL1 occurs at or before the formation of the first peptide bond of MvaL1. Specific interaction of purified MvaL1 with both 23S RNA and its own mRNA is confirmed by filter binding studies. In vivo expression experiments reveal that translation of the distal MvaL10 and MvaL12 cistrons is coupled to that of the MvaL1 cistron. A mRNA secondary structure resembling a canonical L10 binding site and preliminary in vitro regulation experiments had suggested a co‐regulatory function of MvaL10, the homologue of the regulatory protein L10 of the β‐operon of E . coli . However, we show that MvaL10 does not have a regulatory function.

The complete nucleotide sequence of a gene cluster encoding the NusG and the L 11-L1-L10-L12 ribosomal proteins from the thermophilic crenarchaeon Sulfolobus solfataricus has been determined. The genes are arranged in the same order as the equivalent genes in the rif region of Escherichia coli. The ribosomal proteins exhibit between 66% (L10) and 80% (L12) identity with their respective equivalents from Sulfolobus acidocaldarius. The short distance (5 nucleotides) between the nusG stop codon and the L11 start codon suggests that nusG and the genes for the ribosomal proteins are transcribed as a single unit.

An Escherichia coli coupled in vitro transcription-translation system has been modified to allow efficient expression of genes under the control of a T7 promoter. We describe both the characterization and use of two S30 crude extracts prepared from E. coli, namely S30 BL21(DE3) (containing endogenous T7 RNA polymerase) and S30 BL21 (supplemented with exogenous T7 RNA polymerase). Since transcription by the highly active T7 RNA polymerase is known to overload the translational machinery of E. coli, the ratio between mRNA and ribosomes has to be regulated in the coupled in vitro system. For this purpose, the level of mRNA is controlled by varying the amount of DNA template (S30 extract with endogenous T7 RNA polymerase) or by limited amounts of exogenously added T7 RNA polymerase. The coupled in vitro system described in this paper provides two especially useful applications. First, it is most suitable for studying the regulation of gene expression in vitro, second, it can be used to express DNA templates carrying up to 10 genes. We show that genes which are not well expressed in E. coli in vivo because of unfavourable codon usage or plasmid instability are synthesized efficiently in the coupled in vitro system.

The ribosomal protein MvaS7 from the methanogenic archaeon Methanococcus vannielii is a protein of 188 amino acids, i.e., it is 42 amino acids longer than previously suggested. The triplet TTG serves as a start codon. The methanogenic translation initiation region that includes the rare TTG start codon is recognized in Escherichia coli.

The mechanisms for regulation of ribosomal gene expression have been characterized in eukaryotes and eubacteria, but not yet in archaebacteria. We have studied the regulation of the synthesis of ribosomal proteins MvaL1, MvaL10, and MvaL12, encoded by the MvaL1 operon of Methanococcus vannielii, a methanogenic archaebacterium. MvaL1, the homolog of the regulatory protein L1 encoded by the L11 operon of Escherichia coli, was shown to be an autoregulator of the MvaL1 operon. As in E. coli, regulation takes place at the level of translation. The target site for repression by MvaL1 was localized by site-directed mutagenesis to a region within the coding sequence of the MvaL1 gene commencing about 30 bases downstream of the ATG initiation codon. The MvaL1 binding site on the mRNA exhibits similarity in both primary sequence and secondary structure to the L1 regulatory target site of E. coli and to the putative binding site for MvaL1 on the 23S rRNA. In contrast to other regulatory systems, the putative MvaL1 binding site is located in a sequence of the mRNA which is not in direct contact with the ribosome as part of the initiation complex. Furthermore, the untranslated leader sequence is not involved in the regulation. Therefore, we suggest that a novel mechanism of translational feedback regulation exists in M. vannielii.

DEAE‐Sepharose chromatography of extracts from plasmodia of the myxomycete Physarum polycephalum revealed the presence of multiple histone acetyltransferases and histone deacetylases. A cytoplasmic histone acetyltransferase B, specific for histone H4, and two nuclear acetyltransferases A1 and A2 were identified; A1 acetylates all core histones with a preference for H3 and H2A, whereas A2 is specific for H3 and also slightly for H2B. Two histone deacetylases, HD1 and HD2, could be discriminated. They differ with respect to substrate specificity and pH dependence. For the first time the substrate specificity of histone deacetylases was determined using HPLC‐purified individual core histone species. The order of acetylated substrate preference is H2A>H3>‐H4>H2B for HD1 and H3>H2A>H4 for HD2, respectively; HD2 is inactive with H2B as substrate. Moreover histone deacetylases are very sensitive to butyrate, since 2 mM butyrate leads to more than 50% inhibition of enzyme activity.

DEAE-Sepharose chromatography of plasmodial extracts of the myxomycete Physarum polycephalum reveals the presence of multiple histone acetyltransferases and histone deacetylases. Five putative histone acetyltransferases and histone deacetylases. Five putative histone acetyltransferase forms with different substrate specificity can be discriminated: one enzyme which acetylates all core histones and four enzymes with a preference for each of H3, H2A, H2B or H4. Two histone deacetylases, HD1 and HD2, can be discriminated. They differ with respect to substrate specificity and pH-dependence. The substrate specificity of histone deacetylases is determined using HPLC-purified individual core histone species. The order of acetylated substrate preference is H2A>>H3≥H4> H2B for HD1, H3>H2A>H4 for HD2, respectively; HD2 is inactive with H2B as substrate.

Multiple forms of histone acetyltransferases and histone deacetylases, which have been separated and characterized in the accompanying manuscript (Lopez-Rodas, G., Georgieva, E. I., Sendra, R., and Loidl, P. (1991) J. Biol. Chem. 266, 18745-18750), together with in vivo acetate incorporation, were studied during the germination of Zea mays embryos. Total histone acetyltransferase activity increases during germination with two maxima at 40 and 72 h after start of germination. This fluctuation is mainly due to the cytoplasmic B-enzyme which predominantly acetylates histone H4 up to the diacetylated form. The nuclear histone acetyltransferase A2, specific for H3, is low throughout germination, except at 24 h, when it transiently becomes the main activity. Both enzymes are also present in the dry embryo, whereas the second nuclear enzyme A1, specific for H3 and H4, is absent in the initial stage of differentiation. The two histone deacetylases, HD1 and HD2, exhibit entirely different patterns. Whereas HD1 activity is low in the dry embryo and increases during germination, HD2 is the predominant enzyme at the start of differentiation, but almost disappears at later stages. Analysis of the in vivo acetate incorporation reveals that H4 is present in up to tetraacetylated subspecies. The pattern of acetate incorporation into core histones closely resembles the fluctuations of histone acetyltransferase B. Based on the analysis of thymidine kinase activity a close correlation was established between histone acetyltransferase B and DNA replication, whereas the A2 enzyme is associated with transcriptional activity. Histone deacetylase HD1 obviously serves a specific function in the dry embryo and could be a prerequisite for DNA repair processes. The study confirms the idea of multiple functions of histone acetylation and assigns distinct enzymes, involved in this modification, to certain nuclear processes.