Kaichiro Yanagisawa

A new class of rapidly developing mutants in Dictyostelium discoideum: Implications for cyclic AMP metabolism and cell differentiation

Rapidly developing (rde) mutants of Dictyostelium discoideum, in which cells precociously differentiated into stalk and spore cells without normal morphogenesis, were investigated genetically and biochemically. Genetic complementation tests demonstrated that the 16 rde mutants isolated could be classified into at least two groups (groups A and C) and that the first described rde mutant FR17 (D. R. Sonneborn, G. J. White, and M. Sussman, 1963, Dev. Biol. 7, 79-93) belongs to group A. Morphological studies revealed several differences in development and final morphology between group A and group C mutants. In group A mutants, the time required for cell differentiation from vegetative cells to aggregation competent cells is reduced, whereas the time required for spore and stalk cell differentiation following the completion of aggregation is shortened in group C mutants. This suggests that group C mutants represent a new class of rde mutants and that there exist at least two mechanisms involved in regulating the timing of development in D. discoideum. Measurements of cell-associated and extracellular phosphodiesterase activities, and intracellular and total cAMP levels revealed that cAMP metabolism in both groups is significantly altered during development. Group A mutants showed precocious and excessive production of phosphodiesterase and cAMP during the entire course of development; intracellular cAMP levels in group C mutants were extremely low, and spore and stalk cell differentiation occurred without an apparent increase in these levels. Thus, while cAMP metabolism is abnormal in all the rde mutants studied, there exist several distinct types of derangement, not necessarily involving the overproduction of cAMP.

The mitochondrial DNA of Dictyostelium discoideum: Complete sequence, gene content and genome organization

Abstract We present an overview of the gene content and organization of the mitochondrial genome of Dictyostelium discoideum. The mitochondria genome consists of 55,564 bp with an A + T content of 72.6%. The identified genes include those for two ribosomal RNAs (rnl and rns), 18 tRNAs, ten subunits of the NADH dehydrogenase complex (nad1, 2, 3, 4, 4L, 5, 6, 7, 9 and 11), apocytochrome b (cytb), three subunits of the cytochrome oxidase (cox1/2 and 3), four subunits of the ATP synthase complex (atp1, 6, 8 and 9), 15 ribosomal proteins, and five other ORFs, excluding intronic ORFs. Notable features of D. discoideum mtDNA include the following. (1) All genes are encoded on the same strand of the DNA and a universal genetic code is used. (2) The cox1 gene has no termination codon and is fused to the downstream cox2 gene. The 13 genes for ribosomal proteins and four ORF genes form a cluster 15.4 kb long with several gene overlaps. (3) The number of tRNAs encoded in the genome is not sufficient to support the synthesis of mitochondrial protein. (4) In total, five group I introns reside in rnl and cox1/2, and three of those in cox1/2 contain four free-standing ORFs. We compare the genome to other sequenced mitochondrial genomes, particularly that of Acanthamoeba castellanii.

Nuclear fusion in multinucleated giant cells during the sexual development of Dictyostelium discoideum

Abstract Macrocyst development in Dictyostelium discoideum , is generally considered a sexual phase. This development is initiated by the formation of a giant cell, the result of the fusion of two different mating type haploid cells, such as NC4 and HM1. The giant cell engulfs unfused surrounding cells to develop into a macrocyst. Therefore, if the macrocyst is a sexual structure, the giant cell must be a diploid zygote. However, under certain conditions, a very large multinucleated giant cell containing several dozens of nuclei is formed, followed by normal development into a macrocyst. In such a multinucleated giant cell, it was found that only two nuclei fuse together to produce a diploid zygote and all others disappear at the early stage of development. The diploid nucleus undergoes meiosis and subsequently subdivides into a number of haploid progeny cells later released from the macrocyst to initiate new life cycles.

A novel cyclic AMP metabolism exhibited by giant cells and its possible role in the sexual development of Dictyostelium discoideum

In Dictyostelium discoideum cyclic AMP (cAMP) metabolism during macrocyst development, i.e., the sexual cycle of this organism, and in giant cells, i.e., fusion products from opposite mating-type cells, was investigated. The pattern of change in cAMP levels during macrocyst development differed considerably from that observed during fruiting-body formation, i.e., the asexual cycle. Giant cells produced and excreted considerable amounts of cAMP. Adenylate cyclase activity catalyzing cAMP production in giant cells was comparable to that of unfused cells. However, the activity of membrane-bound phosphodiesterase in giant cells was extremely low, and no extracellular phosphodiesterase was excreted. A phosphodiesterase inhibitory protein was secreted in excess by giant cells.

A membrane protein with possible relevance to sexual cell fusion in Dictyostelium discoideum

The molecular mechanism of sexual cell fusion in Dictyostelium discoideum was studied using the heterothallic strains HM1 and NC4. Monovalent antibodies (Fab) prepared from rabbit antiserum against a crude membrane preparation of fusion-competent HM1 cells inhibited fusion between HM1 and NC4 cells. Six out of 43 antigenic proteins were found in fusion-competent HM1 cells but not in fusion-incompetent cells. Among them, only one protein with a molecular mass of 70 kDa was able to neutralize the fusion-inhibiting activity of Fab, suggesting its possible participation in sexual cell fusion.

Environmental factors inducing sexual development inDictyostelium discoideum

In the heterothallic strains NC4 and HM1 ofDictyostelium discoideum, sexual development is initiated by the formation of diploid zygotic giant cells produced through the fusion of these two opposite mating-type haploid cells. For sexual cell fusion, amoeboid cells must first acquire fusion competence, which requires culture under certain environmental conditions, such as darkness, excessive water, and sufficient bacteria as food. However, in the subsequent stages of cell fusion and development of the giant cells into mature macrocysts, cells do not require the above conditions. Cell fusion and development into macrocysts were able to occur even in light with minimum water and in the absence of bacteria. For cell fusion calcium ions were required.

ORF209 of Dictyostelium discoideum mitochondrial DNA has a homologue in chloroplast DNA

From the analysis of rRNA sequences, it is thought that the cellular slime mold Dictyostelium discoideum is one of the earliest organisms to diverge from the mainroute of eukaryote descent [for review 1]. We have interest in the genome organization of the mitochondrial (mt) DNA of this organism from several points of view. While sequencing the 1.7 kb Hind III DNA fragment of the D. discoideum (KAX3) mt DNA which overlapped on the left side of 3.6 kb Pvu I fragment (see Fig. 1 of [2]), we found an open reading frame (ORF209, Fig. A) adopting the universal genetic code. Computer analysis revealed that the ORF has a homologous region at the carboxy-terminus fairly well conserved with tobacco (ORF159) and liverwort Marchantia polyrnorpha (ORF169) chloroplast (ct) genes [3, 4] and cyanobacterium Synechocystis sp. gene (ORF157) [5] (Fig. B). This result prompted us to examine the possible existence of the gene in the

Formation of deletion in Escherichia coli between direct repeats located in the long inverted repeats of a cellular slime mold plasmid: Participation of DNA gyrase

SummaryWe constructed a recombinant plasmid containing the 2.1 kb HindIII fragment of plasmid pDG1, isolated from the cellular slime mold (Dictyostelium sp. strain GA11), and using pAG60 as cloning vector. We found that deletions of the recombinant plasmid took place frequently in Escherichia coli wild-type cells. However, the deletion was not observed when the plasmid was introduced into a strain that was an isogenic temperature-sensitive mutant of the gyrA gene. These results suggest that E. coli DNA gyrase is involved in the mechanisms of the deletion formation. It was shown that the 1.0 kb deletant derived from the 2.1 kb HindIII insert was produced by elimination of a 1.1 kb region. Sequence analysis of the deletants showed that cutting and rejoining took place between two out of the six nearly perfect direct repeats [21 bp palindromic sequences; AAAAAA(T/C)GGC(G/C)GCC(A/G)TTTTTT], located near the distal ends of the inverted repeats, preserving one copy of the repeats. These sequences consist of local short inverted repeats, where cutting and rejoining occur at one of the two regions.

Group-I introns in the cytochrome c oxidase genes of Dictyostelium discoideum : two related ORFs in one loop of a group-I intron, a cox1/2 hybrid gene and an unusually large cox3 gene

Abstract The DNA sequences of cytochrome oxidase (subunits 1, 2 and 3) genes of the cellular slime mold Dictyostelium discoideum mitochondria were determined. The genes for subunits 1 and 2 have a single continuous ORF (COX1/2) which contains four group-I introns. The insertion sites of the two group-I introns (DdOX1/2.2 and DdOX1/2.3) coincide with those of fungal and algal group-I introns, as well as a liverwort group-I intron, in the cytochrome oxidase subunit 1. Interestingly, intron DdOX1/2.2 has two free-standing ORFs in a loop (L8) which have similar amino-acid sequences and are homologous to ai4 DNA endonuclease (I-Sce II) and bi4 RNA maturase found in group-I introns of Saccharomyces cerevisiae mitochondrial DNA. Two group-I introns (DdOX1/2.3 and DdOX1/2.4) also have a free-standing ORF in loop 1 and loop 2, respectively. These results show that these group-I introns and the intronic ORFs have evolved from the same ancestral origin, but that these ORFs have been propagated independently.

Purification and characterization of gp138, a cell surface glycoprotein involved in the sexual cell fusion of Dictyostelium discoideum

Macrocyst formation in Dictyostelium discoideum is initiated by the fusion of cells between two opposite mating-type strains, such as NC-4 and HM1. When these cells become fusion-competent under appropriate environmental conditions, a specific protein involved in sexual cell fusion, 138 kDa, appears on the cell surface of both NC-4 and HM1 strains, as reported previously. The present study was carried out to purify and characterize this protein. The 138 kDa protein was shown to be a glycoprotein (gp138) which binds specifically to lectins, WGA, Con-A or LCA, but not to PNA, PHAE4, RCA60 or RCA120. The isoelectric point of the gp138 was determined as pH 4.5-4.9. To confirm again the previous results, a Coomassie blue-stained gel containing the 138-kDa band was cut out following the SDS-polyacrylamide gel electrophoresis, and antiserum against this band protein(s) was obtained. Fab fragments of this antiserum caused the complete inhibition of sexual fusion between NC-4 and HM1 cells.