Biomedical subjects

Hideko Urushihara

Publications and source records attributed to Hideko Urushihara.

24 records · Page 2Linked to original sources

PubMed2003

Construction of a gamete-enriched gene pool and RNAi-mediated functional analysis in Dictyostelium discoideum.

Macrocysts in Dictyostelium discoideum possess prototypic features of sexual reproduction and are useful for understanding the basic mechanisms of the reproductive process. Here, we randomly analyzed 1,071 gamete cDNAs, and then constructed a gamete-specific subtraction library, FC-IC. Nucleotide sequences of all 903 FC-IC clones were determined and clustered into 272 independent genes. Expression analysis based on real-time RT-PCR revealed 67 gamete-enriched genes, among which those involved in 'signal transduction' and 'multicellular organization' are prevalent. One of them, FC-IC0003, appeared also to be mating-type specific, and was named gmsA. RNAi-mediated silencing as well as disruption of gmsA reduced the cellular competency for sexual cell fusion, indicating the involvement of this gene in the sexual development of D. discoideum.

Amino Acid Sequence↗

PubMed2003

Changing patterns of gene expression in dictyostelium prestalk cell subtypes recognized by in situ hybridization with genes from microarray analyses.

We used microarrays carrying most of the genes that are developmentally regulated in Dictyostelium to discover those that are preferentially expressed in prestalk cells. Prestalk cells are localized at the front of slugs and play crucial roles in morphogenesis and slug migration. Using whole-mount in situ hybridization, we were able to verify 104 prestalk genes. Three of these were found to be expressed only in cells at the very front of slugs, the PstA cell type. Another 10 genes were found to be expressed in the small number of cells that form a central core at the anterior, the PstAB cell type. The rest of the prestalk-specific genes are expressed in PstO cells, which are found immediately posterior to PstA cells but anterior to 80% of the slug that consists of prespore cells. Half of these are also expressed in PstA cells. At later stages of development, the patterns of expression of a considerable number of these prestalk genes changes significantly, allowing us to further subdivide them. Some are expressed at much higher levels during culmination, while others are repressed. These results demonstrate the extremely dynamic nature of cell-type-specific expression in Dictyostelium and further define the changing physiology of the cell types. One of the signals that affect gene expression in PstO cells is the hexaphenone DIF-1. We found that expression of about half of the PstO-specific genes were affected in a mutant that is unable to synthesize DIF-1, while the rest appeared to be DIF independent. These results indicate that differentiation of some aspects of PstO cells can occur in the absence of DIF-1.

Animals↗

PubMed2002

Functional genomics of the social amoebae, Dictyostelium discoideum.

Dictyostelium discoideum is one of the simplest organisms to form a multicellular structure, and it offers several advantages as a model. In order to understand the genetic basis of the multicellular development, a comprehensive analysis of cDNAs is being performed. To date, about 75,000 ESTs have been collected at different stages of development. They have been assembled into about 6,400 independent sequences that represent 70-80% of all of the expected genes in D. discoideum. The results are available on the Internet. In addition to structural analyses, functional analyses of the temporal and spatial expression patterns and gene targeting are being carried out. Furthermore, there are plans to combine the information that is obtained from the cDNA, Genome, and Proteome Projects, as well as the published results, into an integrated database, DictyBase.

Animals↗

PubMed2002

A putative serpentine receptor gene tasA required for normal morphogenesis of primary stalk and branch structure in Polysphondylium pallidum.

The fruiting body of Polysphondylium pallidum is composed of whorls of branches along the axis of a primary stalk. In the course of fruiting body formation, the interval between neighboring whorls and the number and the spacing of branches in a whorl are highly regulated. In this study, using restriction enzyme mediated integration mutagenesis, we have obtained a mutant (strain M6226) with thicker and aberrant primary stalk. The gene responsible for the mutant phenotype, confirmed by homologous recombination, encodes an open reading frame with 383 aa residues (46.3 kDa) and was named thick and aberrant stalk A (tasA). TasA is highly homologous to Dictyostelium discoideum cyclic adenosine 3',5'-monophosphate receptors. A tasA transcript is expressed strictly at the late aggregation stage. Cells expressing a tasA::gfp fusion DNA are localized at the posterior region of the primary sorogen where secondary sorogens and branches originate. This result indicates the existence of 'prebranch' and 'pretrunk' regions in P. pallidum instead of the prespore and prestalk regions in D. discoideum. The analyzes of the gene disruptant and chimeric fruiting bodies also suggests that TasA affects the normal morphogenesis of the primary stalk and the process of cell differentiation into prebranch cells, but not into spore or stalk cells directly.

Amino Acid Sequence↗

PubMed2002

PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors.

We introduce a PCR-based procedure for generating a gene disruption construct. This method depends on DNA fragment fusion by the PCR technique and requires only two steps of PCR to obtain a sufficient amount of the gene disruption construct for one transformation experiment. The first step involves three separate PCR syntheses of a selectable marker cassette and the 5'- and 3'-regions of a target gene. Of the four primers used in amplification of the 5'- and 3'-regions of the target gene, two primers placed proximal to the site of the marker cassette are designed to have sequence tags complementary to the 5'- or 3'-side of the marker cassette. The two primers used in PCR synthesis of the marker cassette are complementary to the tagged primers. By fusion PCR, the 5' and 3' PCR products are linked to the marker cassette via the regions of tagged primers that overlap. A sufficient amount of the disruption construct can be directly amplified with the outermost primers. This method is simple, rapid and relatively inexpensive. In addition, there is the freedom of attaching long flanking regions to any selectable marker cassette.

Alleles↗

PubMed2002

A transcriptional profile of multicellular development in Dictyostelium discoideum.

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 regulated during development.

Animals↗