Local variability of the phosphoglycerate kinase-triosephosphate isomerase fusion protein from Thermotoga maritima MSB 8.

The pgk-tpi gene locus of Thermotoga maritima encodes both phosphoglycerate kinase (PGK) and a bienzyme complex consisting of a fusion protein of PGK with triosephosphate isomerase (TIM). No separate tpi gene for TIM is present in T. maritima. A frame-shift at the end of the pgk gene has been previously proposed as a mechanism to regulate the expression of the two protein variants [Schurig et al., EMBO J. 14 (1995), 442-451]. Surprisingly, the complete T. maritima genome was found to contain a pgk-tpi sequence not requiring the proposed frameshift mechanism. To clarify the apparent discrepancy, a variety of DNA sequencing techniques were applied, disclosing an anomalous local variability in the pgk-tpi fusion region. The comparison of different DNA samples and the mass spectrometric analysis of the amino acid sequence of the natural fusion protein from T. maritima MSB8 confirmed the local variability of the DNA variants. Since not all peptide masses could be assigned, further variations are conceivable, suggesting an even higher heterogeneity of the T. maritima MSB8 strain.

Intraspecies analysis: comparison of ITS sequence data and gene intron sequence data with breeding data for a worldwide collection of Gonium pectorale.

The morphologically uniform species Gonium pectorale is a colonial green flagellate of worldwide distribution. The affinities of 25 isolates from 18 sites on five continents were assessed by both DNA sequence comparisons and sexual compatibility. Complete sequences were obtained (i) for the internal transcribed spacer ITS-1 and ITS-2 regions of ribosomal DNA and (ii) for each of three single-copy spliceosomal introns, two in a small G protein and one in the actin gene. ITS sequences appeared to homogenize sufficiently rapidly to behave as a single copy gene. Intron sequence differences between isolates in this species reached nucleotide substitution saturation, while ITS sequences did not. Parsimony and evolutionary distance analysis of the two types of DNA data gave essentially the same tree conformation. By all these criteria, the group of G. pectorale isolates fell into two main clades, A and B. Clade A, with isolates from four continents, was comprised of four subclades of quite closely related isolates, plus one strain of ambiguous affinity. Clade B was comprised of two subclades represented by South African and South American isolates, respectively; thus, only subclades of clade B showed geographical localization. With respect to mating, all isolates except one homothallic strain and one apparently sterile strain fell into either one or the other of two mating types. Pairings in all possible combinations revealed that isolates from the same site formed abundant zygotes, which germinated to produce new, sexually active organisms. Zygotes were also formed in many pairings of other combinations, including crosses of clade A with clade B organisms, but none of the latter produced viable germlings. The ability to mate and produce viable progeny that were themselves capable of sexual reproduction was restricted to members of subclades established on the basis of DNA sequence similarities. Thus, the grades of difference in both nuclear intron sequences and rDNA ITS sequences paralleled those observed in the sexual analysis.

Inhibition of germ tube formation, filamentation and ergosterol biosynthesis in Candida albicans treated with 6-amino-2-n-pentylthiobenzothiazole.

Three representative Candida albicans strains were selected out of 26 clinical isolates and strains from culture collections on the basis of their high level of conversion to germ tubes and mycelial form at mycelium-promoting culture conditions, and on their different sensitivity to 6-amino-2-n-pentylthiobenzothiazole (APB). When these strains were treated with APB at mycelium-promoting culture conditions, a concentration-dependent decrease in the proportion of germ tubes and hyphae was observed, while the proportion of the yeast from increased. When non-saponifiable lipids were extracted from these cultures and analyzed, a concentration-dependent decrease in ergosterol and an increase in 4-methylated sterols was observed. However, the sensitivity of sterol biosynthesis did not directly relate to the sensitivity of the morphological conversion, and was exhibited at higher concentrations of APB. On the basis of these results it is suggested that the inhibition of germ tube formation and filamentation is not a consequence of inhibition of ergosterol biosynthesis in APB-treated C. albicans.

Brefeldin A affects synthesis and integrity of a eukaryotic flagellum.

Eukaryotic flagella and cilia are highly dynamic organelles. In green algae like Chlamydomonas reinhardtii, flagella absorption and resynthesis is a normal process during the vegetative cell cycle. Rapid regeneration also occurs after stress-induced shedding of flagella. Ca2+ ions, protein synthesis, and a kinase activity are the main factors known to affect resynthesis. Recently, we have detected that certain small G proteins (Ypt/Rab) and a GTPase regulator (GDP dissociation inhibitor), known as regulatory elements of intracellular vesicle transport, are present in flagellar membranes of green algae, raising the possibility that the organelle's synthesis and/or integrity depends on functional membrane traffic. In this study, we examined the effect of brefeldin A (BFA), an inhibitor of intracellular membrane flow and Golgi function in animal and plant cells, on flagella regeneration in the colonial green alga Gonium pectorale. We show that low BFA concentrations (< 1 microgram/ml) inhibit flagella out-growth, while higher amounts cause dose-dependent deflagellation and cell death. Our findings provide experimental evidence for a direct connection between intracellular transport and eukaryotic flagella synthesis.

Rapid evolution of sex-related genes in Chlamydomonas.

Biological speciation ultimately results in prezygotic isolation-the inability of incipient species to mate with one another-but little is understood about the selection pressures and genetic changes that generate this outcome. The genus Chlamydomonas comprises numerous species of unicellular green algae, including numerous geographic isolates of the species C. reinhardtii. This diverse collection has allowed us to analyze the evolution of two sex-related genes: the mid gene of C. reinhardtii, which determines whether a gamete is mating-type plus or minus, and the fus1 gene, which dictates a cell surface glycoprotein utilized by C. reinhardtii plus gametes to recognize minus gametes. Low stringency Southern analyses failed to detect any fus1 homologs in other Chlamydomonas species and detected only one mid homolog, documenting that both genes have diverged extensively during the evolution of the lineage. The one mid homolog was found in C. incerta, the species in culture that is most closely related to C. reinhardtii. Its mid gene carries numerous nonsynonymous and synonymous codon changes compared with the C. reinhardtii mid gene. In contrast, very high sequence conservation of both the mid and fus1 sequences is found in natural isolates of C. reinhardtii, indicating that the genes are not free to drift within a species but do diverge dramatically between species. Striking divergence of sex determination and mate recognition genes also has been encountered in a number of other eukaryotic phyla, suggesting that unique, and as yet unidentified, selection pressures act on these classes of genes during the speciation process.

Intron sequences provide a tool for high-resolution phylogenetic analysis of volvocine algae.

Three nuclear spliceosomal introns in conserved locations were amplified and sequenced from 28 strains representing 14 species and 4 genera of volvocalean green algae. Data derived from the three different introns yielded congruent results in nearly all cases. In pairwise comparisons, a spectrum of taxon-specific sequence differences ranging from complete identity to no significant similarity was observed, with the most distantly related organisms lacking any conserved elements apart from exon-intron boundaries and a pyrimidine-rich stretch near the 3' splice site. A metric (SI50), providing a measure of the degree of similarity of any pair of intron sequences, was defined and used to calculate phylogenetic distances between organisms whose introns displayed statistically significant similarities. The rate of sequences divergence in the introns was great enough to provide useful information about relationships among different geographical isolates of a single species, but in most cases was too great to provide reliable guides to relationships above the species level. A substitution rate of approximately 3 x 10(-8) per intron position per year was estimated, which is about 150-fold higher than in nuclear genes encoding rRNA and about 10-fold higher than the synonymous substitution rate in protein-coding regions. Thus, these homologous introns not only provide useful information about intraspecific phylogenetic relationships, but also illustrate the concept that different parts of a gene may be subject to extremely different intensities of selection. The intron data generated here (1) reliably resolve for the first time the relationships among the five most extensively studied strains of Volvox, (2) reveal that two other Volvox species may be more closely related than had previously been suspected, (3) confirm prior evidence that particular isolates of Eudorina elegans and Pleodorina illinoisensis appear to be sibling taxa, and (4) contribute to the resolution of several hitherto unsettled issues in Chlamydomonas taxonomy.

Identification and characterization of a lower plant Ypt/Rab guanosine dissociation inhibitor (GDI).

The cDNA encoding a Ypt/Rab guanosine dissociation inhibitor (Ypt-GDI) was isolated from the multicellular green alga Volvox carteri, representing the first complete plant gdi gene described. The gdiV1 gene occurs as a single copy in the algal genome, indicating that its product regulates all YptV proteins from Volvox. The derived GDI protein (GDIV1p) shows high similarity to animal and fungal GDIs. A specific antibody developed against GDIV1p detected the protein throughout the whole Volvox life-cycle. GDIV1p was localized in the cytoplasm and in the algal flagellum. This is in line with earlier findings of a dual localization of Ypt proteins both in the cell body and in the motility organelle, and indicates a novel role of the GDI/Ypt system, possibly in intraflagellar transport.

Small G proteins of two green algae are localized to exocytic compartments and to flagella.

The Ypt/Rab proteins are small GTPases, which belong to the Ras superfamily and have been shown to be involved in endo- and exocytosis in mammalian cells and yeast. Using affinity-purified antibodies specific for four Ypt proteins, namely Ypt1p, Ypt4p, Ypt5p and Ypt6p, of the multicellular green alga Volvox carteri (YptVp) and its close unicellular relative Chlamydomonas reinhardtii (YptCp), we examined the abundance of the corresponding antigens during the asexual life cycle of Volvox, and their intracellular localization. The YptV proteins were found in all stages throughout the asexual life cycle and are tightly associated with intracellular membranes. Indirect immunofluorescence revealed that YptV4p, YptV5p and YptV6p are present in perinuclear regions of the cell, indicating an association with the Golgi region. Golgi localization of YptV4p and YptV6p in Volvox was confirmed by immunogold electron microscopy. In contrast, we found Ypt1p associated with the contractile vacuole in both V. carteri and C. reinhardtii. Furthermore, the YptV proteins were also detected along the entire length of the flagella of somatic Volvox cells. This flagellar location was substantiated by western blot analysis of extracts prepared from isolated flagella of both algae. While localization to exocytic compartments is in agreement with the established Ypt/Rab function in intracellular vesicle transport of eukaryotic cells, presence in the algal flagellum is the first hint of a possible role for small G proteins also in motility organelles.

Tomato Rab1A homologs as molecular tools for studying Rab geranylgeranyl transferase in plant cells.

Rab proteins attach to membranes along the secretory pathway where they contribute to distinct steps in vesicle-mediated transport. To bind membranes, Rab proteins in fungal and animal cells must be isoprenylated by the enzyme Rab geranylgeranyl transferase (Rab GGTase). We have isolated three tomato (Lycopersicon esculentum, M.) cDNAs (LeRab 1A, B, and C) encoding Rab-like proteins and show here that all three are substrates for a Rab GGTase-like activity in plant cells. The plant enzyme is similar to mammalian Rab GGTase in that the plant activity (a) is enhanced by detergent and (b) is inhibited by mutant Rab lacking a prenylation consensus sequence. LeRab1B contains a rare prenylation target motif and was the best substrate for the plant, but not the yeast, Rab GGTase. LeRab1A, B, and C are functional homologs of the Saccharomyces cerevisiae Rab protein encoded by YPT1 and are differentially expressed in tomato. LeRab1A mRNA, but not that of LeRab1B or C, is induced by ethylene in tomato seedlings and is also upregulated in ripening fruit. The increase in LeRab1A mRNA expression in ripe fruit may be linked to increased synthesis and export of enzymes like polygalacturonase, pectin esterase, and other enzymes important in fruit softening.

The organization structure and regulatory elements of Chlamydomonas histone genes reveal features linking plant and animal genes.

The genome of the green alga Chlamydomonas reinhardtii contains approximately 15 gene clusters of the nucleosomal (or core) histone H2A, H2B, H3 and H4 genes and at least one histone H1 gene. Seven non-allelic histone gene loci were isolated from a genomic library, physically mapped, and the nucleotide sequences of three isotypes of each core histone gene species and one linked H1 gene determined. The core histone genes are organized in clusters of H2A-H2B and H3-H4 pairs, in which each gene pair shows outwardly divergent transcription from a short (< 300 bp) intercistronic region. These intercistronic regions contain typically conserved promoter elements, namely a TATA-box and the three motifs TGGCCAG-G(G/C)-CGAG, CGTTGACC and CGGTTG. Different from the genes of higher plants, but like those of animals and the related alga Volvox, the 3' untranslated regions contain no poly A signal, but a palindromic sequence (3' palindrome) essential for mRNA processing is present. One single H1 gene was found in close linkage to a H2A-H2B pair. The H1 upstream region contains the octameric promoter element GGTTGACC (also found upstream of the core histone genes) and two specific sequence motifs that are shared only with the Volvox H1 promoters. This suggests differential transcription of the H1 and the core histone genes. The H1 gene is interrupted by two introns. Unlike Volvox H3 genes, the three sequenced H3 isoforms are intron-free. Primer-directed PCR of genomic DNA demonstrated, however, that at least 8 of the about 15 H3 genes do contain one intron at a conserved position. In synchronized C. reinhardtii cells, H4 mRNA levels (representative of all core histone mRNAs) peak during cell division, suggesting strict replication-dependent gene control. The derived peptide sequences place C. reinhardtii core histones closer to plants than to animals, except that the H2A histones are more animal-like. The peptide sequence of histone H1 is closely related to the V. carteri VH1-II (66% identity). Organization of the core histone gene in pairs, and non-polyadenylation of mRNAs are features shared with animals, whereas peptide sequences and enhancer elements are shared with higher plants, assigning the volvocalean histone genes a position intermediate between animals and plants.

Analysis of a family of ypt genes and their products from Chlamydomonas reinhardtii.

Small G-proteins encoded by the ras-like ypt genes are ubiquitous in eukaryotic cells. They have been shown to play an essential role in membrane vesicle transport. We have isolated four ypt genes, yptC1, yptC4, yptC5 and yptC6, from Chlamydomonas reinhardtii (Cr) genomic and cDNA libraries. Three of them, yptC1, yptC4 and yptC5, are close homologues of ypt genes previously found in the multicellular alga Volvox carteri (Vc), the fourth, yptC6, is new. Each yptC gene is present as a single copy in the genome. Comparisons of genomic and cDNA sequences revealed that the coding regions are interrupted by five (yptC5), six (yptC6), seven (yptC4) and eight (yptC1) introns, respectively. Cr ypt genes and the closely related Vc ypt genes have identical exon-intron structures, but the corresponding intron sequences are completely different. Polyadenylation is signalled by UAUAA, UGUAG and UGUAA. The deduced amino acid (aa) sequence of YptC6 exhibited 79% identity with HRab2; YptC1, YptC4 and YptC5 exhibited over 90% identity with their Vc homologues. Primary structures of the 9-aa 'effector domain' and the contiguous 'helix3-loop7' motif (approx. 30 aa) are 'diagnostic' features for functional assignment. Recombinant YptC proteins, overproduced in Escherichia coli and purified to near homogeneity, displayed strong and specific binding of GTP, but not of GMP or ATP. The four Cr Ypt proteins showed immunochemical cross reactions to their Vc counterparts. Moreover, Western blots demonstrated at least six types of Ypt in both Cr and Vc, suggesting that these Ypt are used for household functions responsible for vesicle transport rather than for cellular differentiation.

Structure-function analysis of small G proteins from Volvox and Chlamydomonas by complementation of Saccharomyces cerevisiae YPT/SEC mutations.

cDNAs representing nine small G protein genes encoding Ypt proteins from the green algae Volvox carteri (YptV) and Chlamydomonas reinhardtii (YptC) were tested for their ability to complement mutations in the YPT1, SEC4, and YPT7 genes of Saccharomyces cerevisiae strains defective in different steps of intracellular vesicle transport. None of the heterologously expressed algal genes was able to complement mutations in SEC4 or YPT7, but three of them, yptV1, yptC1, and yptV2, restored a YPT1 null mutation. On the amino acid sequence level, and particularly with respect to known small G protein specificity domains, YptV1p and YptC1p are the closest algal analogs of yeast Ypt1p, with 70% overall identity and identical effector regions, but YptV2p is only 55% identical to Ypt1p, and its effector domain resembles that of Sec4p. To define more precisely the regions that supply Ypt1p function, six chimeras were constructed by reciprocal exchange of 68/72-, 122/123-, and 162/163-amino acid segments of the C-terminal regions between YptV1p (complementing) and YptV3p (non-complementing). Segments containing 68 amino acids of the hypervariable C-terminal, and 41 residues of the N-terminal region including the effector region, of YptV1p could be replaced by the corresponding parts of YptV3p without loss of function in yeast, but exchanges within the central core destroyed the ability to rescue the YPT1 mutation. Sequence analysis of ypt1-complementing and -noncomplementing Ypt types suggests that surface loop3 represents a novel specificity domain of small G proteins.

5-Aminosalicylic acid abrogates T-cell proliferation by blocking interleukin-2 production in peripheral blood mononuclear cells.

The antiinflammatory agent sulfasalazine (SS) is prescribed to treat Crohn's disease, ulcerative colitis and rheumatoid arthritis. Activated T cells are present within diseased mucosal and synovial sites. We tested whether SS or its metabolites 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP) inhibited the T-cell activation products interleukin 2 (IL-2) and interleukin 2 receptor alpha-chain (IL-2R alpha). Experiments were performed in phytohemaglutinin- and phorbol ester-stimulated peripheral blood mononuclear cells. Radioactive thymidine and leucine incorporation assayed DNA and protein synthesis, respectively. Enzyme-linked immunosorbent assay and Northern blot analysis measured IL-2 and IL-2R alpha. Lactate dehydrogenase release determined cell viability, and intracellular free calcium was measured by an indole fluorescent indicator. SS and 5-ASA, but not SP, inhibited T-cell proliferation and protein synthesis in phytohemaglutinin- and phorbol ester-stimulated peripheral blood monomuclear cells. 5-ASA (625 microM) markedly reduced culture supernatant IL-2 protein levels by 92% and steady-state IL-2 messenger RNA levels 4.4-fold at 24 and 18 hr, respectively. The supplementation of IL-2 restored T-cell proliferation only in 5-ASA-treated cultures. SS, 5-ASA and SP did not alter intracellular calcium accumulation after mitogenic stimulation. SS and 5-ASA (625 microM) caused 71% and 37% cytotoxicity, respectively, in 72-hr cultures. 5-ASA inhibits T-cell proliferation in part by blocking IL-2 messenger RNA accumulation and protein production downstream of the rise in cytosolic calcium. Inhibition of IL-2 production is an additional mechanism of action for 5-ASA.

Analysis of the introns in genes encoding small G proteins.

Because all small G proteins (SGPs) possess a very similar array of structural and functional domains, they are obvious candidates for examining the relationships postulated to exist between the exon-intron structure of genes and the domain structure of the encoded proteins. To address this issue, and to possibly gain insight into the evolution of their introns, we have analyzed positions, sizes, and sequences of 125 introns from 28 SGP genes. These introns were found to be distributed in 60 different locations throughout the aligned sequences, with a preference for the 5'-half of the genes. More than 50% of the positions were found to be shared by two or more genes, and genes encoding SGPs of very similar amino acid sequence (i.e., isotypes) in quite closely related species tend to have most, or all, of their introns in identical locations, indicating a common evolutionary origin (homologous introns). However, with few exceptions, no statistically significant sequence similarity or common folding motif was found between homologous intron pairs. Only three intron positions are shared between members of distantly related SGP subfamilies. These three potentially ancient intron locations fall between regions encoding alpha-helices or beta-sheets, but two of them interrupt regions encoding known functional (guanosine-nucleotide-binding) modules. Intron positions that are occupied only in single genes, or in genes encoding very similar SGPs, do not show any preferential distribution with respect to regions encoding structural or functional motifs.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure, expression, and phylogenetic relationships of a family of ypt genes encoding small G-proteins in the green alga Volvox carteri.

In addition to the previously described gene yptV1 encoding a small G-protein we have now identified and sequenced four more ras-related ypt genes (yptV2-yptV5) from the green alga Volvox carteri. The four new genes encode polypeptides consisting of 203 to 217 amino-acid residues that contain the typical sequence elements (GTP-binding domains, effector domain) of the ypt/rab subgroup of the Ras superfamily. Comparison of the derived amino-acid sequences from the V. carteri ypt gene products and their Ypt homologs from other species revealed similarity values ranging from 60% to 85%, whereas intraspecies similarities were found to approach only 55%. The coding sequences are interrupted by 5-7 introns of variable size (70-1000 nucleotides) occupying different positions in the genes. Reverse-transcribed samples of stage-specific RNAs were PCR-amplified with primers specific to yptV1, yptV3, yptV4, and yptV5 to determine if yptV transcription might be restricted to either cell type or to a specific stage of the life cycle. These experiments demonstrated that each of these genes is expressed throughout the entire Volvox life cycle and in both the somatic and the reproductive cells of the alga. The transcription start sites of yptV1 and yptV5 were mapped by primer extension. Expression of recombinant yptV cDNA in E. coli yielded recombinant proteins that bound GTP specifically, demonstrating a property which is typical for small G-proteins. The derived YptV polypeptide sequences were used to group them into four distinct classes of Ras-like proteins. These are the first proteins of the Ras superfamily to be identified in a green alga. We discuss the possible role of the YptV-proteins in the intracellular vesicle transport of Volvox.

The yptV1 gene encodes a small G-protein in the green alga Volvox carteri: gene structure and properties of the gene product.

Small G-proteins encoded by ras-like genes are ubiquitous in eukaryotic cells. These G-proteins are believed to play a role in central processes, such as signal transduction, cell differentiation and membrane vesicle transport. By screening genomic and cDNA libraries of the colonial alga, Volvox carteri f. nagariensis, with ypt DNA probes from Zea mays, we have identified the first member of a ypt gene family, yptV1, within a green alga. The 1538-bp yptV1 gene of V. carteri consists of nine exons and eight introns and has three potential polyadenylation sites 210, 420 and 500 bp downstream from the UGA stop codon. The derived 203-amino-acid polypeptide, YptV1, exhibits 81% similarity with Ypt1 from mouse, with the corresponding genes sharing four identical intron positions. Recombinant YptV1 (reYptV1) produced in Escherichia coli retains the ability to bind GTP after SDS-PAGE and immobilization on nitrocellulose. Immunological studies using polyclonal antibodies against reYptV1 indicate that the protein is present in the membrane fraction of a V. carteri extract and is expressed throughout the whole life-cycle of the alga. Similar to other Ras-like proteins, YptV1 contains two conserved C-terminal cysteine residues suggesting post-translational modification(s), such as isoprenylation or palmitoylation, required for membrane anchoring. The presumptive role of YptV1 in cytoplasmic vesicle transport is briefly discussed.

Analysis and nucleotide sequence of the genes encoding the surface-layer glycoproteins of the hyperthermophilic methanogens Methanothermus fervidus and Methanothermus sociabilis.

The genes (slgA) encoding the surface-layer glycoproteins of the hyperthermophilic methanogens Methano-thermus pervidus and Methanothermus sociabilis were cloned and sequenced. The nucleotide sequences of these genes differ at only nine positions, resulting in three amino acid differences. In both organisms, the transcription start site was localized by primer extension analyses. The DNA sequence at this site conforms to the promotor box B motif for promotors of archaea. 24 nucleotides upstream of the transcription start is an A + T-rich region, which closely resembles the consensus box A motif of promoters of methanogens. Ribosome binding sites are exactly complementary to the 3' end of the 16S rRNA of these methanogens. Both slgA genes encode for a precursor of the mature surface-layer protein containing 593 amino acid residues with a putative N-terminal signal sequence of 22 amino acid residues. The deduced protein sequences contain 20 sequon structures representing possible carbohydrate-binding sites. In comparison with other surface-layer proteins, these obtained from the two hyperthermophilic methanogens contain unusually high amounts of isoleucine, asparagine and cysteine residues. Predicted secondary structures have a high content of beta-sheet structure (44%) and only 7% alpha-helix structures.