scholarly journals A prespore-cell-inducing factor in Dictyostelium discoideum: its purification and characterization

1999 ◽  
Vol 343 (1) ◽  
pp. 265-271 ◽  
Author(s):  
Manabu NAKAGAWA ◽  
Akiko A. OOHATA ◽  
Hiromasa TOJO ◽  
Shigeru FUJII
Keyword(s):  
Cell Division ◽  
Cell Differentiation ◽  
Amino Acid ◽  
Conditioned Medium ◽  
Dictyostelium Discoideum ◽  
Periodic Acid ◽  
Limited Proteolysis ◽  
Amino Acid Sequences ◽  
Homology Search ◽  
Prespore Cell

Under starvation conditions, amoebae of Dictyostelium discoideum aggregate to form multicellular masses; the aggregates are then initiated to differentiate. We have reported previously that a signal substance exists in conditioned medium of D. discoideum, and we named it prespore-cell-inducing factor (psi, Ψ factor) [Oohata, Nakagawa, Tasaka, and Fujii (1997) Development 124, 2781-2787]. The factor can induce isolated amoebae to differentiate into prespore cells. Moreover, we suggested that it caused not only cell differentiation but also cell division. In the present study, we have purified Ψ factor from the conditioned medium and characterized it. The purified Ψ factor induced both prespore cell differentiation and cell division of prespore cells. Its apparent molecular mass was 180 kDa by gel filtration and 106 kDa by SDS/PAGE. Based on these results, Ψ factor exists as a dimer in normal conditions. Periodic acid/Schiff staining showed that Ψ factor was a glycoprotein. It was ascertained by Edman degradation that Ψ factor is blocked at the N-terminal. Treatment with pyroglutamate aminopeptidase removed the N-terminal block and allowed determination of the amino-acid sequence of Ψ factor. Moreover, three internal amino-acid sequences were determined in limited proteolysis experiments using trypsin and endoproteinase Lys-C. The homology search for these sequences supports the fact that Ψ factor is a novel differentiation factor.

scholarly journals A novel prespore-cell-inducing factor in Dictyostelium discoideum induces cell division of prespore cells

Development ◽  
1997 ◽  
Vol 124 (14) ◽  
pp. 2781-2787 ◽  
Author(s):  
A.A. Oohata ◽  
M. Nakagawa ◽  
M. Tasaka ◽  
S. Fujii
Keyword(s):  
Cell Division ◽  
Cell Differentiation ◽  
Dictyostelium Discoideum ◽  
Gel Filtration ◽  
Relative Molecular Mass ◽  
Heat Stable ◽  
Prespore Cell ◽  
Sds Page ◽  
Cell Induction ◽  
Low Cell Density

In Dictyostelium discoideum strain V12M2, at a very low cell density (approximately 10(2) cells/cm2), most amoebae differentiate into prespore cells in a salt solution containing cAMP if an adequately diluted conditioned medium (CM) is provided (Oohata, A. A. (1995) Differentiation 59, 283–288). This finding suggests the presence of factor(s) released into the medium that are involved in inducing prespore cell differentiation. In the present study, we report the presence of two types of factors that function synergistically in prespore cell induction; one is a heat-stable and dialysable factor(s) and the other is a heat-labile and non-dialysable factor termed psi (psi) factor (prespore-inducing factor). We purified and characterized the psi factor. Its relative molecular mass was determined to be 106x10(3) Mr by SDS-PAGE and 180x10(3) Mr by gel filtration HPLC, respectively. These results indicate that psi factor exists as a dimer under native conditions. In addition to inducing prespore cell differentiation, psi factor induced cell division of prespore cells in submerged culture. Our results suggest that psi factor plays important roles not only in prespore cell differentiation but also in the progress of the cell cycle in the prespore pathway in normal development.

102 PROTEOMIC ANALYSIS OF CONDITIONED MEDIUM SUPPLEMENTED WITH PORCINE FOLLICULAR FLUID

2011 ◽  
Vol 23 (1) ◽  
pp. 156
Author(s):  
S. Hwang ◽  
K. B. Oh ◽  
H.-C. Lee ◽  
B.-C. Yang ◽  
D. Lim ◽  
...  
Keyword(s):  
Conditioned Medium ◽  
Follicular Fluid ◽  
Culture Medium ◽  
Cell Metabolism ◽  
Eukaryotic Cell ◽  
Amino Acid Sequences ◽  
Homology Search ◽  
Surface Binding ◽  
Culture Methods

Follicular fluid (FF) contains growth factors, electrolytes, hormones, amino acids, and unknown factors. Supplementation of porcine FF (pFF) to in vitro maturation (IVM) medium was reported to improve the oocyte maturation, monospermic fertilization and embryonic development. This study aimed at investigating whether pFF supplementation affects the characteristics of donor cells for somatic cell nuclear transfer and the proteomic composition of the culture medium. Ear fibroblast cells from an NIH major histocompatibility complex (MHC) inbred miniature pig were cultured with different culture methods: 1) DMEM + 10% FBS (FBS); 2) DMEM + 10% FBS + 10% pFF (pFF). The conditioned medium was collected at 72 h. After isoelectric focusing (IEF), the equilibrated strips were submitted to SDS-PAGE. Normalized protein spots were considered significantly different between the two groups if expression levels varied by two standard deviations. To identify the protein spots, an Ettan MALDI-TOF method was used. Upon submission of the amino acid sequences, proteins were identified by a homology search using ProteinInfo or BLAST search using the ExPASy Molecular Biology Server. The proportion of G0/G1 stage cells in the pFF group was significantly higher than the proportions in the other groups (P < 0.05). Among 42 differentially expressed spots, 36 proteins were identified in the pFF group. Some molecular functions of the spots were: catalytic or methytransferase activity, eukaryotic cell surface binding, or ferric iron binding. It can be concluded that pFF supplementation of culture medium positively affects cell-cycle synchronization and cell metabolism. Further studies are needed to analyse the function of these important cellular proteins. This work received grant support from the Agenda Program (No. PJ006688) and (No. PJ007189), Rural Development Administration, Republic of Korea.

Isolation and characterization of the pea cytochrome c oxidase Vb gene

Genome ◽  
2006 ◽  
Vol 49 (11) ◽  
pp. 1481-1489 ◽  
Author(s):  
Nakao Kubo ◽  
Shin-ichi Arimura ◽  
Nobuhiro Tsutsumi ◽  
Koh-ichi Kadowaki ◽  
Masashi Hirai
Keyword(s):  
Amino Acid ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Fluorescent Proteins ◽  
Amino Acid Sequences ◽  
Homology Search ◽  
Coding Region ◽  
Angiosperm Evolution ◽  
Isolation And Characterization ◽  
Duplication Events

Three copies of the gene that encodes cytochrome c oxidase subunit Vb were isolated from the pea (PscoxVb-1, PscoxVb-2, and PscoxVb-3). Northern Blot and reverse transcriptase-PCR analyses suggest that all 3 genes are transcribed in the pea. Each pea coxVb gene has an N-terminal extended sequence that can encode a mitochondrial targeting signal, called a presequence. The localization of green fluorescent proteins fused with the presequence strongly suggests the targeting of pea COXVb proteins to mitochondria. Each pea coxVb gene has 5 intron sites within the coding region. These are similar to Arabidopsis and rice, although the intron lengths vary greatly. A phylogenetic analysis of coxVb suggests the occurrence of gene duplication events during angiosperm evolution. In particular, 2 duplication events might have occurred in legumes, grasses, and Solanaceae. A comparison of amino acid sequences in COXVb or its counterpart shows the conservation of several amino acids within a zinc finger motif. Interestingly, a homology search analysis showed that bacterial protein COG4391 and a mitochondrial complex I 13 kDa subunit also have similar amino acid compositions around this motif. Such similarity might reflect evolutionary relationships among the 3 proteins.

The role of diffusible molecules in regulating the cellular differentiation of Dictyostelium discoideum

Development ◽  
1988 ◽  
Vol 103 (1) ◽  
pp. 1-16 ◽  
Author(s):  
J.G. Williams
Keyword(s):  
Developmental Biology ◽  
Cell Differentiation ◽  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Cellular Differentiation ◽  
Prespore Cell ◽  
Additional Role ◽  
Acting In Concert ◽  
Morphogenetic Fields

A central problem in developmental biology is to understand how morphogenetic fields are created and how they act to direct regionalized cellular differentiation. This goal is being pursued in organisms as diverse as moulds, worms, flies, frogs and mice. Each organism has evolved its own solution to the challenge of multicellularity but there appear to be common underlying principles and, once pattern formation is fully understood in any system, some general truths seem certain to be revealed. As a non-obligate metazoan, Dictyostelium discoideum has proven a particularly tractable system in which to identify and characterize cellular morphogens. Cyclic AMP and ammonia stimulate prespore cell differentiation and ammonia plays an additional role in repressing terminal cellular differentiation. Differentiation Inducing Factor (DIF) acts to direct prestalk cell differentiation and adenosine may play a synergistic role in repressing prespore cell differentiation. This review summarizes the evidence for these interactions and describes a number of models which show how this small repertoire of diffusible molecules, acting in concert, may direct the formation of a differentiated structure.

Dictyostelium discoideum contains two profilin isoforms that differ in structure and function

1991 ◽  
Vol 100 (3) ◽  
pp. 481-489 ◽  
Author(s):  
M. Haugwitz ◽  
A.A. Noegel ◽  
D. Rieger ◽  
F. Lottspeich ◽  
M. Schleicher
Keyword(s):  
Amino Acid ◽  
Cdna Library ◽  
Dictyostelium Discoideum ◽  
Actin Polymerization ◽  
Critical Concentration ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Actin Binding ◽  
Gene Coding ◽  
And Function

Two profilin isoforms (profilins I and II) have been purified from Dictyostelium discoideum, using affinity chromatography on a poly(L-proline) matrix; the isoforms could be separated by cation-exchange chromatography on a FPLC system. The gene coding for profilin I was cloned from a lambda gt11 cDNA library using a profilin I-specific monoclonal antibody. The profilin II cDNA was isolated by probing the cDNA library with an oligonucleotide deduced from the N-terminal amino acid sequence of profilin II, which has an open N terminus in contrast to profilin I. The deduced amino acid sequences of both genes show that profilin I in comparison to profilin II is slightly larger (13,064 Da vs 12,729 Da), has a more acidic isoelectric point (calc. pI 6.62 vs 7.26) and shares with profilin II 68 identical residues out of 126 amino acids. Although both profilins contain a conserved lysine residue in the putative actin-binding region and can be crosslinked covalently to G-actin, the crosslinking efficiency of profilin II to actin is substantially higher than that of profilin I. These data are in agreement with studies on the functional properties of the profilin isoforms. In most preparations profilin II was more efficient in delaying the onset of elongation during the course of actin polymerization and caused a higher critical concentration for actin polymerization than profilin I, probably due to the slightly increased affinity of profilin II for D. discoideum G-actin (approx. Kd 1.8 × 10(−6) M) as compared to that of profilin I (approx. Kd 5.1 × 10(−6) M).(ABSTRACT TRUNCATED AT 250 WORDS)

LlaFI, a Type III Restriction and Modification System in Lactococcus lactis

1999 ◽  
Vol 65 (2) ◽  
pp. 686-693 ◽  
Author(s):  
Ping Su ◽  
Heejeong Im ◽  
Hsiaoling Hsieh ◽  
Simon Kang’A ◽  
Noel W. Dunn
Keyword(s):  
Amino Acid ◽  
Lactococcus Lactis ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Homology Search ◽  
Binding Motif ◽  
Significant Similarity ◽  
Modification System ◽  
Type Iii ◽  
Central Regions

ABSTRACT We describe a type III restriction and modification (R/M) system,LlaFI, in Lactococcus lactis. LlaFI is encoded by a 12-kb native plasmid, pND801, harbored in L. lactisLL42-1. Sequencing revealed two adjacent open reading frames (ORFs). One ORF encodes a 680-amino-acid polypeptide, and this ORF is followed by a second ORF which encodes an 873-amino-acid polypeptide. The two ORFs appear to be organized in an operon. A homology search revealed that the two ORFs exhibited significant similarity to type III restriction (Res) and modification (Mod) subunits. The complete amino acid sequence of the Mod subunit of LlaFI was aligned with the amino acid sequences of four previously described type III methyltransferases. Both the N-terminal regions and the C-terminal regions of the Mod proteins are conserved, while the central regions are more variable. An S-adenosyl methionine (Ado-Met) binding motif (present in all adenine methyltransferases) was found in the N-terminal region of the Mod protein. The seven conserved helicase motifs found in the previously described type III R/M systems were found at the same relative positions in the LlaFI Res sequence.LlaFI has cofactor requirements for activity that are characteristic of the previously described type III enzymes. ATP and Mg2+ are required for endonucleolytic activity; however, the activity is not strictly dependent on the presence of Ado-Met but is stimulated by it. To our knowledge, this is the first type III R/M system that has been characterized not just in lactic acid bacteria but also in gram-positive bacteria.

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