By using monoclonal antibodies directed against discoidin II, we have isolated cDNA clones from a... more By using monoclonal antibodies directed against discoidin II, we have isolated cDNA clones from axenically grown Ax-2 cells. One cDNA clone (D2) contained a 1.2-kb insert encoding the entire discoidin II protein, which is composed of 257 amino acid residues and has a calculated molecular mass of 28,574. The amino acid sequences, determined by Edman degradation of six tryptic peptides of discoidin II, were identical to those deduced from the cDNA sequence. The protein bears no resemblance to any proteins in the data banks, except that its sequence is 49% identical with the amino acid sequence of discoidin I. Discoidin II shares with discoidin I both a carbohydratebinding site and an Arg-Gly-Asp (RGD) sequence, which has been found in nbronectin in mammalian cells. With the onset of aggregation (8 h of development), a 1.3-kb discoidin II mRNA begins to accumulate. A similar pattern of regulation occurs at the protein level.
Tunicamycin acts on cell aggregation in Dictyostelium discoideum by changing cell movement and by... more Tunicamycin acts on cell aggregation in Dictyostelium discoideum by changing cell movement and by inhibiting the EDTA-stable type of intercellular adhesion. Tunicamycin- treated cells show unco-ordinated pseudopodial activity such that pseudopods are simultaneously extended from all parts of the cell surface, and the cells are unable to move in straight paths. Concurrent with the inhibition of formation of EDTA-stable contacts, N-glycosylation of a glycoprotein specific for aggregation-competent cells is inhibited. This glycoprotein, previously called contact site A, has an apparent mol. wt. of 80 kilodaltons (kd). In membranes of tunicamycin-treated cells, two components are detected that react with certain monoclonal antibodies against contact sites A: one component of 66 kd, the other of 53 kd apparent mol. wt. Another group of monoclonal antibodies reacts only with the 80-kd glycoprotein and the 66-kd component. These results are in accord with the assumption that the glycoprotein carries two carbohydrate chains, and that the antibodies differ in their requirement for glycosylation of the antigen. Despite the coincidence between blockage of EDTA-stable cell adhesion and inhibited glycosylation of contact sites A, direct involvement of the carbohydrate moieties of this glycoprotein in intercellular adhesion seems questionable. EDTA-stable cell adhesion has not been blocked by Fab fragments from anti- bodies that specifically react with the glycosylated protein.
The gp64 protein of Polysphondylium pallidum has been shown to mediate EDTA-stable cellcell adhes... more The gp64 protein of Polysphondylium pallidum has been shown to mediate EDTA-stable cellcell adhesion. To explore the functional role of gp64, we made an antisense RNA expression construct designed to prevent the gene expression of gp64; the construct was introduced into P. pallidum cells and the transformants were characterised. The antisense RNA-expressing clone L3mc2 which had just been harvested at the growth phase tended to reform in aggregates smaller in size than did the parental cells in either the presence or absence of 10 mM EDTA. In contrast, 6.5-hour starved L3mc2 cells remained considerably dissociated from each other after 5 minutes gyrating, although aggregation gradually increased by 50% during a further 55 minutes gyrating, in the presence of 10 mM EDTA. Correspondingly, L3mc2 lacked specifically the cellcell adhesion protein, gp64. We therefore conclude that the gp64 protein is involved in forming the EDTA-resistant cellcell contact. In spite of the absence of gp64,...
A distinct feature of development in the simple eukaryote Dictyostelium discoideum is an aggregat... more A distinct feature of development in the simple eukaryote Dictyostelium discoideum is an aggregative transition from a unicellular to a multicellular phase. Using genome-wide transcriptional analysis we show that this transition is accompanied by a dramatic change in the expression of more than 25% of the genes in the genome. We also show that the transcription patterns of these genes are not sensitive to the strain or the nutritional history, indicating that Dictyostelium development is a robust physiological process that is accompanied by stereotypical transcriptional events. Analysis of the two differentiated cell types, spores and stalk cells, and their precursors revealed a large number of differentially expressed genes as well as unexpected patterns of gene expression, which shed new light on the timing and possible mechanisms of cell-type divergence. Our findings provide new perspectives on the complexity of the developmental program and the fraction of the genome that is reg...
Acetoacetyl-CoA thiolase (Acat) is an enzyme that catalyzes both the CoA-dependent thiolytic clea... more Acetoacetyl-CoA thiolase (Acat) is an enzyme that catalyzes both the CoA-dependent thiolytic cleavage of acetoacetyl-CoA and the reverse condensation reaction. In Dictyostelium disciodeum, acetoacetyl-CoA thiolase (DdAcat) is encoded by a single acat gene. The aim of this study was to assess the localization of DdAcat and to determine the mechanism of its cellular localization. Subcellular localization of DdAcat was investigated using its fusion protein with the green fluorescent protein (GFP), and it was found to be localized to peroxisomes. The findings showed that the targeting signal of DdAcat to peroxisomes is a unique nonapeptide sequence (15RMYTTAKNL23) similar to the conserved peroxisomal targeting signal-2 (PTS-2). Cell fractionation experiments revealed that DdAcat also exists in the cytosol. Distribution in the cytosol was caused by translational initiation from the second Met codon at position 16. The first 18 N-terminal residues also exhibited function as a mitochondria...
A cDNA with homology to fatty acid desaturases was selected by searching the cDNA data bank of Di... more A cDNA with homology to fatty acid desaturases was selected by searching the cDNA data bank of Dictyostelium discoideum (http://www.csm.biol.tsukuba.ac.jp/cDNAproject.html) with conserved histidine box motifs. Using this sequence, genomic DNA encoding the D5 desaturase was amplified from the genomic DNA of D. discoideum, and its desaturase activity was confirmed by the overexpression mutation in D. discoideum and the gain-of-function mutation in yeast. The cloned cDNA is 1565 nucleotides in length, and the deduced aminoacid sequence comprised 467 amino-acid residues containing an N-terminal cytochrome b5 domain that shared 43% identity with cytochrome b5 of Oryza sativa. The whole sequence was 42% identical to the D5 desaturase of Mortierella alpina. This desaturase is a novel member of the cytochrome b5-containing D5 fatty acid desaturase. As we have already reported one other D5 desaturase in Dictyostelium, this organism is the first to be confirmed as having two functional D5 fatty acid desaturase genes. The substrate specificities of the two functional D5 desaturases of D. discoideum were also examined.
The cellular slime mold, Polysphondylium pallidum, has two EDTA-resistant types of cell-cell adhe... more The cellular slime mold, Polysphondylium pallidum, has two EDTA-resistant types of cell-cell adhesion. The major component of them has been identified as a glycoprotein with a molecular mass of 64 kDa on SDS-PAGE (referred to as gp64). We found that a substantial amount of the gp64 run as dimer, when gp64 was dissolved in SDS-sample buffer without 2-mercaptoethanol and then subjected to electrophoresis. The occurrence of a homophilic dimer was demonstrated by analyzing the dimer-like band on a gel for its amino acid sequence and amino acid composition. The dimer-like band also was analyzed by three sorts of monoclonal antibodies, two of which recognize respectively a conformational epitope and a denatured epitope of the protein moiety of gp64. The data indicate that the native conformation of gp64 is necessary for dimer formation.
Dictyostelium is a favored model for studying problems in cell and developmental biology. To comp... more Dictyostelium is a favored model for studying problems in cell and developmental biology. To comprehend the genetic potential and networks that direct growth and multicellular development, we are performing a large-scale analysis of Dictyostelium cDNAs. Here, we newly determine 7720 nucleotide sequences of cDNAs from the multicellular, slug stage (S) and 10 439 from the unicellular, vegetative stage (V). The combined 26 954 redundant ESTs were computer assembled using the PHRAP program to yield 5381 independent sequences. These 5381 predicted genes represent about half of the estimated coding potential of the organism. One-third of them were classi®ed into 12 functional categories. Although the overall classi®cation patterns of the V and S libraries were very similar, stage-speci®c genes exist in every category. The majority of V-speci®c genes function in some aspect of protein translation, while such genes are in a minority in the S-speci®c and common populations. Instead, genes for signal transduction and multicellular organization are enriched in the population of S-speci®c genes. Genes encoding the enzymes of basic metabolism are mainly found in the common gene population. These results therefore suggest major differences between growing and developing Dictyostelium cells in the nature of the genes transcribed.
In an effort to identify and characterize genes expressed during multicellular development in Dic... more In an effort to identify and characterize genes expressed during multicellular development in Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNA from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, library S, consisting of 9984 clones, carries relatively short inserts, and the other, library L, which consists of 8448 clones, has longer inserts. We sequenced all the selected clones in library S from their 3'-ends, and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs hit known Dictyostelium genes and 910 showed significant similarity to genes of Dictyostelium and other organisms. For library L, 1132 clones were randomly sequenced and 471 non-redundant ESTs were obtained. In combination, the ESTs from the two libraries represent approximately 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will provide a useful resource for investigating the genetic networks that regulate multicellular development of this organism.
We analyzed the spatial expression patterns of the genes involved in myosin function by in situ h... more We analyzed the spatial expression patterns of the genes involved in myosin function by in situ hybridization at the tipped aggregate and early culmination stages of Dictyostelium. Myosin heavy chain II mRNA was enriched in the anterior prestalk region of the tipped aggregates, whereas it disappeared from there and began to appear in both upper and lower cups of the early culminants. Similarly, mRNAs for essential light chain, regulatory light chain, myosin light chain kinase A, and myosin heavy chain kinase C were enriched in the prestalk region of the tipped aggregates. However, expression of these genes was distinctively regulated in the early culminants. These findings suggest the existence of mechanisms responsible for the expression of particular genes.
β-Oxidation of long-chain fatty acids and branched-chain fatty acids is carried out in mammalian ... more β-Oxidation of long-chain fatty acids and branched-chain fatty acids is carried out in mammalian peroxisomes by a multifunctional enzyme (MFE) or d -bifunctional protein, with separate domains for hydroxyacyl coenzyme A (CoA) dehydrogenase, enoyl-CoA hydratase, and steroid carrier protein SCP2. We have found that Dictyostelium has a gene, mfeA , encoding MFE1 with homology to the hydroxyacyl-CoA dehydrogenase and SCP2 domains. A separate gene, mfeB , encodes MFE2 with homology to the enoyl-CoA hydratase domain. When grown on a diet of bacteria, Dictyostelium cells in which mfeA is disrupted accumulate excess cyclopropane fatty acids and are unable to develop beyond early aggregation. Axenically grown mutant cells, however, developed into normal fruiting bodies composed of spores and stalk cells. Comparative analysis of whole-cell lipid compositions revealed that bacterially grown mutant cells accumulated cyclopropane fatty acids that remained throughout the developmental stages. Suc...
cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desatura... more cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) information of the Dictyostelium cDNA project. Using this sequence, we have determined the cDNA sequence and genomic sequence of a desaturase. The cloned cDNA is 1489 nucleotides long and the deduced amino acid sequence comprised 464 amino acid residues containing an N-terminal cytochrome b5 domain. The whole sequence was 38.6% identical to the initially identified Delta5-desaturase of Mortierella alpina. We have confirmed its function as Delta5-desaturase by over expression mutation in D. discoideum and also the gain of function mutation in the yeast Saccharomyces cerevisiae. Analysis of the lipids from transformed D. discoideum and yeast demonstrated the accumulation of Delta5-desaturated products. This is the first report concering fatty acid desaturase in cellular slime molds.
Membrane fluidity is critical for proper membrane function and is regulated in part by the propor... more Membrane fluidity is critical for proper membrane function and is regulated in part by the proportion of unsaturated fatty acids present in membrane lipids. The proportion of these lipids in turn varies with temperature and may contribute to temperature adaptation in poikilothermic organisms. The fundamental question posed in this study was whether the unsaturation of fatty acids contributes to the ability to adapt to temperature stress inDictyostelium. First, fatty acid composition was analysed and it was observed that the relative proportions of dienoic acids changed with temperature. To investigate the role of dienoic fatty acids in temperature adaptation, null mutants were created in the two known Δ5 fatty acid desaturases (FadA and FadB) that are responsible for the production of dienoic fatty acids. ThefadBnull mutant showed no significant alteration in fatty acid composition or in phenotype. However, the disruption offadAresulted in a large drop in dienoic fatty acid content ...
Dictyostelium discoideum has protein kinases AKT/PKBA and PKBR1 that belong to the AGC family of ... more Dictyostelium discoideum has protein kinases AKT/PKBA and PKBR1 that belong to the AGC family of kinases. The protein kinase B-related kinase (PKBR1) has been studied with emphasis on its role in chemotaxis, but its roles in late development remained obscure. The pkbR1 null mutant stays in the first finger stage for about 16 h or longer. Only a few aggregates continue to the migrating slug stage; however, the slugs immediately go back probably to the previous first finger stage and stay there for approximately 37 h. Finally, the mutant fingers diversify into various multicellular bodies. The expression of the pkbR1 finger protein probably is required for development to the slug stage and to express ecmB , which is first observed in migrating slugs. The mutant also showed no ST-lacZ expression, which is of the earliest step in differentiation to one of the stalk cell subtypes. The pkbR1 null mutant forms a small number of aberrant fruiting bodies, but in the presence of 10% of wild-t...
We cloned and characterized a cDNA encoding the Dictyostelium discoideum beta subunit of mitochon... more We cloned and characterized a cDNA encoding the Dictyostelium discoideum beta subunit of mitochondrial processing peptidase (Ddbeta-MPP). Western blot analysis of the mitochondrial subfractions revealed that Ddbeta-MPP is located in the mitochondrial matrix and membrane, whereas Dd(alpha)-MPP, another subunit of DdMPP, is located only in the matrix. Although expression of Ddbeta-MPP mRNA is down-regulated during early development, the level of the Ddbeta-MPP protein is constant throughout the Dictyostelium life cycle. In a transformant expressing the antisense RNA of the beta-MPP gene, unexpectedly, the beta-MPP protein increased about 1.8-fold relative to the wild type, and its mRNA increased 4.5-fold. Expression of other mitochondrial proteins, alpha-MPP and Cox IV, was also induced. These results suggest that antisense RNA inhibition of the beta-MPP gene induces gene expression of mitochondrial proteins, presumably in a retrograde signaling manner. This is the pathway of the transfer of information from the mitochondria to the nucleus.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1999
We cloned a genomic fragment of the membrane protein gp64 gene of the cellular slime mold Polysph... more We cloned a genomic fragment of the membrane protein gp64 gene of the cellular slime mold Polysphondylium pallidum by inverse PCR. Primer extension analysis identified a major transcription start site 65 bp upstream of the translation start codon. The promoter region of the gp64 gene contains sequences homologous to a TATA box at position 347 to 337 and to an initiator (Inr, PyPyCAPyPyPyPy) at position 33 to +5 from the transcription start site. Successively truncated segments of the promoter were tested for their ability to drive expression of the L-galactosidase reporter gene in transformed cells; also the difference in activity between growth conditions was compared. The results indicated that there are two positive vegetative regulatory elements extending between 3187 and 362 bp from the transcription start site of the gp64 promoter; also their activity was two to three times higher in the cells grown with bacteria in shaken suspension than in the cells grown in an axenic medium.
By using monoclonal antibodies directed against discoidin II, we have isolated cDNA clones from a... more By using monoclonal antibodies directed against discoidin II, we have isolated cDNA clones from axenically grown Ax-2 cells. One cDNA clone (D2) contained a 1.2-kb insert encoding the entire discoidin II protein, which is composed of 257 amino acid residues and has a calculated molecular mass of 28,574. The amino acid sequences, determined by Edman degradation of six tryptic peptides of discoidin II, were identical to those deduced from the cDNA sequence. The protein bears no resemblance to any proteins in the data banks, except that its sequence is 49% identical with the amino acid sequence of discoidin I. Discoidin II shares with discoidin I both a carbohydratebinding site and an Arg-Gly-Asp (RGD) sequence, which has been found in nbronectin in mammalian cells. With the onset of aggregation (8 h of development), a 1.3-kb discoidin II mRNA begins to accumulate. A similar pattern of regulation occurs at the protein level.
Tunicamycin acts on cell aggregation in Dictyostelium discoideum by changing cell movement and by... more Tunicamycin acts on cell aggregation in Dictyostelium discoideum by changing cell movement and by inhibiting the EDTA-stable type of intercellular adhesion. Tunicamycin- treated cells show unco-ordinated pseudopodial activity such that pseudopods are simultaneously extended from all parts of the cell surface, and the cells are unable to move in straight paths. Concurrent with the inhibition of formation of EDTA-stable contacts, N-glycosylation of a glycoprotein specific for aggregation-competent cells is inhibited. This glycoprotein, previously called contact site A, has an apparent mol. wt. of 80 kilodaltons (kd). In membranes of tunicamycin-treated cells, two components are detected that react with certain monoclonal antibodies against contact sites A: one component of 66 kd, the other of 53 kd apparent mol. wt. Another group of monoclonal antibodies reacts only with the 80-kd glycoprotein and the 66-kd component. These results are in accord with the assumption that the glycoprotein carries two carbohydrate chains, and that the antibodies differ in their requirement for glycosylation of the antigen. Despite the coincidence between blockage of EDTA-stable cell adhesion and inhibited glycosylation of contact sites A, direct involvement of the carbohydrate moieties of this glycoprotein in intercellular adhesion seems questionable. EDTA-stable cell adhesion has not been blocked by Fab fragments from anti- bodies that specifically react with the glycosylated protein.
The gp64 protein of Polysphondylium pallidum has been shown to mediate EDTA-stable cellcell adhes... more The gp64 protein of Polysphondylium pallidum has been shown to mediate EDTA-stable cellcell adhesion. To explore the functional role of gp64, we made an antisense RNA expression construct designed to prevent the gene expression of gp64; the construct was introduced into P. pallidum cells and the transformants were characterised. The antisense RNA-expressing clone L3mc2 which had just been harvested at the growth phase tended to reform in aggregates smaller in size than did the parental cells in either the presence or absence of 10 mM EDTA. In contrast, 6.5-hour starved L3mc2 cells remained considerably dissociated from each other after 5 minutes gyrating, although aggregation gradually increased by 50% during a further 55 minutes gyrating, in the presence of 10 mM EDTA. Correspondingly, L3mc2 lacked specifically the cellcell adhesion protein, gp64. We therefore conclude that the gp64 protein is involved in forming the EDTA-resistant cellcell contact. In spite of the absence of gp64,...
A distinct feature of development in the simple eukaryote Dictyostelium discoideum is an aggregat... more A distinct feature of development in the simple eukaryote Dictyostelium discoideum is an aggregative transition from a unicellular to a multicellular phase. Using genome-wide transcriptional analysis we show that this transition is accompanied by a dramatic change in the expression of more than 25% of the genes in the genome. We also show that the transcription patterns of these genes are not sensitive to the strain or the nutritional history, indicating that Dictyostelium development is a robust physiological process that is accompanied by stereotypical transcriptional events. Analysis of the two differentiated cell types, spores and stalk cells, and their precursors revealed a large number of differentially expressed genes as well as unexpected patterns of gene expression, which shed new light on the timing and possible mechanisms of cell-type divergence. Our findings provide new perspectives on the complexity of the developmental program and the fraction of the genome that is reg...
Acetoacetyl-CoA thiolase (Acat) is an enzyme that catalyzes both the CoA-dependent thiolytic clea... more Acetoacetyl-CoA thiolase (Acat) is an enzyme that catalyzes both the CoA-dependent thiolytic cleavage of acetoacetyl-CoA and the reverse condensation reaction. In Dictyostelium disciodeum, acetoacetyl-CoA thiolase (DdAcat) is encoded by a single acat gene. The aim of this study was to assess the localization of DdAcat and to determine the mechanism of its cellular localization. Subcellular localization of DdAcat was investigated using its fusion protein with the green fluorescent protein (GFP), and it was found to be localized to peroxisomes. The findings showed that the targeting signal of DdAcat to peroxisomes is a unique nonapeptide sequence (15RMYTTAKNL23) similar to the conserved peroxisomal targeting signal-2 (PTS-2). Cell fractionation experiments revealed that DdAcat also exists in the cytosol. Distribution in the cytosol was caused by translational initiation from the second Met codon at position 16. The first 18 N-terminal residues also exhibited function as a mitochondria...
A cDNA with homology to fatty acid desaturases was selected by searching the cDNA data bank of Di... more A cDNA with homology to fatty acid desaturases was selected by searching the cDNA data bank of Dictyostelium discoideum (http://www.csm.biol.tsukuba.ac.jp/cDNAproject.html) with conserved histidine box motifs. Using this sequence, genomic DNA encoding the D5 desaturase was amplified from the genomic DNA of D. discoideum, and its desaturase activity was confirmed by the overexpression mutation in D. discoideum and the gain-of-function mutation in yeast. The cloned cDNA is 1565 nucleotides in length, and the deduced aminoacid sequence comprised 467 amino-acid residues containing an N-terminal cytochrome b5 domain that shared 43% identity with cytochrome b5 of Oryza sativa. The whole sequence was 42% identical to the D5 desaturase of Mortierella alpina. This desaturase is a novel member of the cytochrome b5-containing D5 fatty acid desaturase. As we have already reported one other D5 desaturase in Dictyostelium, this organism is the first to be confirmed as having two functional D5 fatty acid desaturase genes. The substrate specificities of the two functional D5 desaturases of D. discoideum were also examined.
The cellular slime mold, Polysphondylium pallidum, has two EDTA-resistant types of cell-cell adhe... more The cellular slime mold, Polysphondylium pallidum, has two EDTA-resistant types of cell-cell adhesion. The major component of them has been identified as a glycoprotein with a molecular mass of 64 kDa on SDS-PAGE (referred to as gp64). We found that a substantial amount of the gp64 run as dimer, when gp64 was dissolved in SDS-sample buffer without 2-mercaptoethanol and then subjected to electrophoresis. The occurrence of a homophilic dimer was demonstrated by analyzing the dimer-like band on a gel for its amino acid sequence and amino acid composition. The dimer-like band also was analyzed by three sorts of monoclonal antibodies, two of which recognize respectively a conformational epitope and a denatured epitope of the protein moiety of gp64. The data indicate that the native conformation of gp64 is necessary for dimer formation.
Dictyostelium is a favored model for studying problems in cell and developmental biology. To comp... more Dictyostelium is a favored model for studying problems in cell and developmental biology. To comprehend the genetic potential and networks that direct growth and multicellular development, we are performing a large-scale analysis of Dictyostelium cDNAs. Here, we newly determine 7720 nucleotide sequences of cDNAs from the multicellular, slug stage (S) and 10 439 from the unicellular, vegetative stage (V). The combined 26 954 redundant ESTs were computer assembled using the PHRAP program to yield 5381 independent sequences. These 5381 predicted genes represent about half of the estimated coding potential of the organism. One-third of them were classi®ed into 12 functional categories. Although the overall classi®cation patterns of the V and S libraries were very similar, stage-speci®c genes exist in every category. The majority of V-speci®c genes function in some aspect of protein translation, while such genes are in a minority in the S-speci®c and common populations. Instead, genes for signal transduction and multicellular organization are enriched in the population of S-speci®c genes. Genes encoding the enzymes of basic metabolism are mainly found in the common gene population. These results therefore suggest major differences between growing and developing Dictyostelium cells in the nature of the genes transcribed.
In an effort to identify and characterize genes expressed during multicellular development in Dic... more In an effort to identify and characterize genes expressed during multicellular development in Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNA from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, library S, consisting of 9984 clones, carries relatively short inserts, and the other, library L, which consists of 8448 clones, has longer inserts. We sequenced all the selected clones in library S from their 3'-ends, and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs hit known Dictyostelium genes and 910 showed significant similarity to genes of Dictyostelium and other organisms. For library L, 1132 clones were randomly sequenced and 471 non-redundant ESTs were obtained. In combination, the ESTs from the two libraries represent approximately 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will provide a useful resource for investigating the genetic networks that regulate multicellular development of this organism.
We analyzed the spatial expression patterns of the genes involved in myosin function by in situ h... more We analyzed the spatial expression patterns of the genes involved in myosin function by in situ hybridization at the tipped aggregate and early culmination stages of Dictyostelium. Myosin heavy chain II mRNA was enriched in the anterior prestalk region of the tipped aggregates, whereas it disappeared from there and began to appear in both upper and lower cups of the early culminants. Similarly, mRNAs for essential light chain, regulatory light chain, myosin light chain kinase A, and myosin heavy chain kinase C were enriched in the prestalk region of the tipped aggregates. However, expression of these genes was distinctively regulated in the early culminants. These findings suggest the existence of mechanisms responsible for the expression of particular genes.
β-Oxidation of long-chain fatty acids and branched-chain fatty acids is carried out in mammalian ... more β-Oxidation of long-chain fatty acids and branched-chain fatty acids is carried out in mammalian peroxisomes by a multifunctional enzyme (MFE) or d -bifunctional protein, with separate domains for hydroxyacyl coenzyme A (CoA) dehydrogenase, enoyl-CoA hydratase, and steroid carrier protein SCP2. We have found that Dictyostelium has a gene, mfeA , encoding MFE1 with homology to the hydroxyacyl-CoA dehydrogenase and SCP2 domains. A separate gene, mfeB , encodes MFE2 with homology to the enoyl-CoA hydratase domain. When grown on a diet of bacteria, Dictyostelium cells in which mfeA is disrupted accumulate excess cyclopropane fatty acids and are unable to develop beyond early aggregation. Axenically grown mutant cells, however, developed into normal fruiting bodies composed of spores and stalk cells. Comparative analysis of whole-cell lipid compositions revealed that bacterially grown mutant cells accumulated cyclopropane fatty acids that remained throughout the developmental stages. Suc...
cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desatura... more cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) information of the Dictyostelium cDNA project. Using this sequence, we have determined the cDNA sequence and genomic sequence of a desaturase. The cloned cDNA is 1489 nucleotides long and the deduced amino acid sequence comprised 464 amino acid residues containing an N-terminal cytochrome b5 domain. The whole sequence was 38.6% identical to the initially identified Delta5-desaturase of Mortierella alpina. We have confirmed its function as Delta5-desaturase by over expression mutation in D. discoideum and also the gain of function mutation in the yeast Saccharomyces cerevisiae. Analysis of the lipids from transformed D. discoideum and yeast demonstrated the accumulation of Delta5-desaturated products. This is the first report concering fatty acid desaturase in cellular slime molds.
Membrane fluidity is critical for proper membrane function and is regulated in part by the propor... more Membrane fluidity is critical for proper membrane function and is regulated in part by the proportion of unsaturated fatty acids present in membrane lipids. The proportion of these lipids in turn varies with temperature and may contribute to temperature adaptation in poikilothermic organisms. The fundamental question posed in this study was whether the unsaturation of fatty acids contributes to the ability to adapt to temperature stress inDictyostelium. First, fatty acid composition was analysed and it was observed that the relative proportions of dienoic acids changed with temperature. To investigate the role of dienoic fatty acids in temperature adaptation, null mutants were created in the two known Δ5 fatty acid desaturases (FadA and FadB) that are responsible for the production of dienoic fatty acids. ThefadBnull mutant showed no significant alteration in fatty acid composition or in phenotype. However, the disruption offadAresulted in a large drop in dienoic fatty acid content ...
Dictyostelium discoideum has protein kinases AKT/PKBA and PKBR1 that belong to the AGC family of ... more Dictyostelium discoideum has protein kinases AKT/PKBA and PKBR1 that belong to the AGC family of kinases. The protein kinase B-related kinase (PKBR1) has been studied with emphasis on its role in chemotaxis, but its roles in late development remained obscure. The pkbR1 null mutant stays in the first finger stage for about 16 h or longer. Only a few aggregates continue to the migrating slug stage; however, the slugs immediately go back probably to the previous first finger stage and stay there for approximately 37 h. Finally, the mutant fingers diversify into various multicellular bodies. The expression of the pkbR1 finger protein probably is required for development to the slug stage and to express ecmB , which is first observed in migrating slugs. The mutant also showed no ST-lacZ expression, which is of the earliest step in differentiation to one of the stalk cell subtypes. The pkbR1 null mutant forms a small number of aberrant fruiting bodies, but in the presence of 10% of wild-t...
We cloned and characterized a cDNA encoding the Dictyostelium discoideum beta subunit of mitochon... more We cloned and characterized a cDNA encoding the Dictyostelium discoideum beta subunit of mitochondrial processing peptidase (Ddbeta-MPP). Western blot analysis of the mitochondrial subfractions revealed that Ddbeta-MPP is located in the mitochondrial matrix and membrane, whereas Dd(alpha)-MPP, another subunit of DdMPP, is located only in the matrix. Although expression of Ddbeta-MPP mRNA is down-regulated during early development, the level of the Ddbeta-MPP protein is constant throughout the Dictyostelium life cycle. In a transformant expressing the antisense RNA of the beta-MPP gene, unexpectedly, the beta-MPP protein increased about 1.8-fold relative to the wild type, and its mRNA increased 4.5-fold. Expression of other mitochondrial proteins, alpha-MPP and Cox IV, was also induced. These results suggest that antisense RNA inhibition of the beta-MPP gene induces gene expression of mitochondrial proteins, presumably in a retrograde signaling manner. This is the pathway of the transfer of information from the mitochondria to the nucleus.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1999
We cloned a genomic fragment of the membrane protein gp64 gene of the cellular slime mold Polysph... more We cloned a genomic fragment of the membrane protein gp64 gene of the cellular slime mold Polysphondylium pallidum by inverse PCR. Primer extension analysis identified a major transcription start site 65 bp upstream of the translation start codon. The promoter region of the gp64 gene contains sequences homologous to a TATA box at position 347 to 337 and to an initiator (Inr, PyPyCAPyPyPyPy) at position 33 to +5 from the transcription start site. Successively truncated segments of the promoter were tested for their ability to drive expression of the L-galactosidase reporter gene in transformed cells; also the difference in activity between growth conditions was compared. The results indicated that there are two positive vegetative regulatory elements extending between 3187 and 362 bp from the transcription start site of the gp64 promoter; also their activity was two to three times higher in the cells grown with bacteria in shaken suspension than in the cells grown in an axenic medium.
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Papers by Hiroshi Ochiai