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Query: UNIPROT:P06889 (Mol)
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Physarum polycephalum (strain M3CVIII) contains four unlinked actin gene loci, each with two alleles (ardA1, ardA2, ardB1, ardB2, ardC1, ardC2, ardD1 and ardD2). The 4800 base HindIII fragment of the ardC2 allele was previously isolated as a recombinant phage lambda. We now report the structure of the actin gene sequences (C-actin gene). The gene, which contains four intervening sequences, codes for the principal actin isotype of plasmodia and it is expressed in both the haploid myxamoebal and diploid plasmodial phases of the life cycle. The C-actin isotype is closely related to actins of Dictyostelium, Acanthamoebae, Drosophila, sea urchin and mammalian cytoplasmic actin, and more distantly related to actins of yeast, Entamoebae and Tetrahymena. The ardC1 and ardC2 alleles differ by a 700(+/- 100) base-pair insertion/deletion in the vicinity of the 3' end of the transcribed region of the gene.
J Mol Biol 1988 Jul 05
PMID:Structure and expression of an actin gene of Physarum polycephalum. 317 9

Though actin is ubiquitous in eukaryotes, its existence has not been clearly proven in Tetrahymena. Recently, we have succeeded in cloning and sequencing the Tetrahymena actin gene using a Dictyostelium actin probe (Hirono, M. et al. (1987) J. Mol. Biol. 194, 181-192). The primary structure of the Tetrahymena actin deduced from the nucleotide sequence of its gene is greatly divergent from those of other known actins, making it necessary to ascertain whether the predicted Tetrahymena actin is indeed an actin. In this paper, we investigated the localization of the predicted Tetrahymena actin by an immunofluorescence technique using antibody against its synthetic N-terminal peptide, in order to elucidate its possible biological roles. The results showed that immunofluorescence was localized in the division furrow of the dividing cell, and in the intranuclear filament bundles formed in cells exposed to heat shock or DMSO. In addition, the oral apparatus and the proximity of the cytoproct, which are organelles involved in endocytosis and exocytosis, respectively, also fluoresced. Thus, we conclude that the Tetrahymena actin we identified is indeed an actin and plays the same biological roles as ubiquitous actins do, although it is considerably divergent in its amino acid sequence.
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PMID:Tetrahymena actin: localization and possible biological roles of actin in Tetrahymena cells. 332 92

Several genes which are deactivated on the initiation of development of Dictyostelium discoideum were identified by differential screening of various cDNA libraries. These genes have in common a decrease in the steady-state levels of their corresponding mRNAs on the onset of development and as development proceeds. When development was carried out in the absence of protein synthesis by inhibition with cycloheximide, the decrease in mRNA levels for most genes (V genes) was normal or slightly accelerated. For about 5% of the genes (H genes), however, cycloheximide caused an apparent induction of expression, as revealed by a slight or dramatic increase in mRNA levels, instead of the normal decrease. This effect was due to inhibition of protein synthesis and not to cycloheximide per se. The induction was found to be due to an enhancement of the transcription rate; normal rates of transcription for the H genes were dependent on continued protein synthesis during vegetative growth and development. Thus, two general regulatory classes exist for deactivation of gene expression on initiation of development, one of which is dependent on and one of which is independent of protein synthesis. Analysis of expression of these genes in mutant strains which are aggregation deficient allowed the classes to be subdivided further. Taken together, these characterizations allow several distinct regulatory mechanisms to be identified that are involved in the deactivation of gene expression on the onset of development in D. discoideum.
Mol Cell Biol 1988 Jan
PMID:Effect of protein synthesis inhibition on gene expression during early development of Dictyostelium discoideum. 333 53

We have analyzed the expression of the Dictyostelium gene P8A7 which had been isolated as a cDNA clone from an early developmentally regulated gene. The single genomic copy generated two mRNAs which were subject to different control mechanisms: while one mRNA (P8A7S) was regulated like the cell-type-nonspecific late genes, the other one (P8A7L) was induced during development, when cells were allowed to attach to a substrate, and when cells were subjected to stress, such as heat shock and cadmium. Interestingly the same induction was also observed with cold shock. RNA processing was inhibited by heat and cold shock, leading to nuclear accumulation of a precursor. The translated region of the cDNA was common to both mRNAs and encoded an unusually hydrophobic peptide with the characteristics of a membrane protein.
Mol Cell Biol 1988 Jan
PMID:A developmentally regulated membrane protein gene in Dictyostelium discoideum is also induced by heat shock and cold shock. 333 56

We have constructed and characterized two Dictyostelium transformation vectors (pB10TP1 and pB10TP2) designed for the facile sequence determination, mutagenesis and functional analysis of Dictyostelium genes. The vectors incorporate the B10 neomycin-resistance (neo) gene [Nellen et al., Mol. Cell. Biol. 4 (1984) 2890-2898] and sequences derived pEMBL18+ [Dente et al., Nucl. Acids Res. 11 (1983) 1645-1655], enabling the production of single-stranded template and increasing the yield of double-stranded DNA. A new multiple cloning site (MCS) has been inserted adjacent to the M13 sequence primer binding site so that single-stranded template DNA isolated from recombinants prepared using these vectors is suitable for sequence analysis and site-directed mutagenesis. The linker incorporates restriction sites suitable for the preparation of a directed deletion series and useful in cloning, including some sites with recognition sequences frequent in the extremely A + T-rich Dictyostelium genome. A Dictyostelium genomic fragment has been included to provide transcription termination signals for the neo gene. One of the two vectors (pB10TP1) contains the 3'-proximal portion of a constitutively expressed mRNA of unknown function. It is located downstream from the MCS so that 5'-proximal fragments of genes, cloned into the MCS, generate fusion transcripts which are distinguishable from transcripts of the corresponding endogenous genes. The complete nucleotide sequence of the two vectors has been established and a comprehensive restriction map deduced. We also describe a modification of the published transformation system, which allows it to be applied to the commonly used strain Ax-2, and another generally applicable modification which greatly reduces the time required to obtain stable transformants.
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PMID:Two vectors which facilitate gene manipulation and a simplified transformation procedure for Dictyostelium discoideum. 343 28

We tested the effects of inhibitors of protein and RNA synthesis on the disaggregation-mediated destabilization of prespore mRNAs in Dictyostelium discoideum. Incubating disaggregated cells with daunomycin to inhibit RNA synthesis prevented the loss of prespore mRNAs, whereas the inhibitor decreased or did not affect levels of the common mRNAs CZ22 and actin. Protein synthesis inhibitors varied in their effects. Cycloheximide, which inhibited protein synthesis almost completely, prevented the loss of the prespore mRNAs, but puromycin, which inhibited protein synthesis less well, did not. These results indicate that the process of specific mRNA destabilization requires the synthesis of RNA and possibly of protein.
Mol Cell Biol 1987 Dec
PMID:Specific mRNA destabilization in Dictyostelium discoideum requires RNA synthesis. 343 99

Dictyostelium discoideum synthesizes a 23,000 Mr protein, p23dd-ras, closely related to the mammalian oncogene-encoded protein p21ras. To investigate the subcellular localization of p23dd-ras, conditions were optimized to reduce protein degradation following cell breakage. Subcellular fractionation of D. discoideum showed that p23dd-ras was associated predominantly with the membrane fraction during both vegetative growth and differentiation. In the absence of suitable protease inhibitors considerable amounts of a truncated form of p23dd-ras were recovered in the cytosol fraction, suggesting that intact p23dd-ras is attached to the membrane by a short terminal peptide sequence. Radio-isotope labelling of D. discoideum with myristic acid or palmitic acid in the presence of excess unlabelled acetate resulted in radio-isotope incorporation into a select group of proteins including p23dd-ras. No acyl label appeared in the truncated cytoplasmic form of p23dd-ras when cell breakage was performed in the absence of suitable protease inhibitors, indicating that the acyl group is associated with the short terminal peptide that is cleaved. These data suggest that p23dd-ras, like its mammalian counterpart, is acylated and associated with the plasma membrane. There was no evidence during a 30-minute pulse of methionine for a cytoplasmic precursor to the membrane-bound p23dd-ras, suggesting that the turnover of the presumptive precursor must be much more rapid in D. discoideum than for pro-p21ras in mammalian cells.
Mol Microbiol 1987 Nov
PMID:A ras-encoded protein in Dictyostelium discoideum is acylated and membrane-associated. 344 63

Different wild-type isolates of Dictyostelium discoideum exhibit extensive polymorphism in the length of restriction fragments carrying tRNA genes. These size differences were used to study the organisation of two tRNA gene families which encode a tRNA Val(GUU) and a tRNA Val(GUA) gene. The method used involved a combination of classical D. discoideum parasexual genetics and molecular genetics. The tRNA genes were mapped to specific linkage groups (chromosomes) by correlating the presence of polymorphic DNA bands that hybridized with the tRNA gene probes with the presence of genetic markers for those linkage groups. These analyses established that both of the tRNA gene families are dispersed among sites on several of the chromosomes. Information of nine tRNA Val(GUU) genes from the wild-type isolate NC4 was obtained: three map to linkage group I (C, E, F), two map to linkage group II (D, I), one maps to linkage group IV (G), one, which corresponds to the cloned gene, maps to either linkage group III or VI (B), and two map to one of linkage groups III, VI or VII (A, H). Six tRNA Val(GUA) genes from the NC4 isolate were mapped: one to linkage group I (D), two to linkage group III, VI or VII (B, C) and three to linkage group VII or III (A, E, F).
Mol Gen Genet 1987 Apr
PMID:Chromosomal mapping of tRNA genes from Dictyostelium discoideum. 347 95

We have studied two regions of Dictyostelium discoideum chromatin and identified several DNase I-hypersensitive sites in these regions. One of these sites is located about 300 to 500 bases upstream of the transcriptional start site of a gene that is expressed at all stages of development. This site is present in both vegetative cells and postaggregation cells. Another hypersensitive site is associated with a gene that is expressed only after the multicellular stage. This site is located about 400 bases upstream of the start site, and it is present only in postaggregation cells. Thus, much like higher eucaryotes, D. discoideum contains DNase I-hypersensitive sites that may be involved in the regulation of the genes with which they are associated.
Mol Cell Biol 1987 May
PMID:Developmental regulation of DNase I-hypersensitive sites in Dictyostelium discoideum. 360 Jun 46

Actin is ubiquitous in eukaryotes, nevertheless its existence has not yet been clearly proven in Tetrahymena. Here we report the cloning and sequencing of an actin gene from the genomic library of Tetrahymena pyriformis using a Dictyostelium actin gene as a probe. The Tetrahymena actin gene has no intron. The predicted actin is composed of 375 amino acids like other actins and its molecular weight is estimated as 41,906. Both T. pyriformis and T. thermophila possess a single species of actin genes which differ in their restriction patterns. Northern hybridization analysis revealed that the actin gene was actively transcribed in vivo. To detect the gene product, we synthesized an N-terminal peptide of the deduced sequence and prepared its antibody. Using an immunoblotting technique, we identified Tetrahymena actin on a two-dimensional gel electrophoretic plate. The actin spot migrated near an added spot of rabbit skeletal muscle actin, but clearly differed from the latter in its isoelectric point and apparent molecular weight. The primary structure of Tetrahymena actin shares about 75% homology equally with those of other representative actins. This value is extremely low as a homology rate between known actins. Tetrahymena actin diverges not only in relatively variable regions of other actins, but also in relatively constant regions. The hydrophilicity levels of two regions (residues 190 to 200 and residues 225 to 235) are also quite different between the Tetrahymena actin and skeletal muscle actin. Thus, we conclude that actin is present in Tetrahymena, but it is one of the most unique actins among the actins known hereto.
J Mol Biol 1987 Mar 20
PMID:Tetrahymena actin. Cloning and sequencing of the Tetrahymena actin gene and identification of its gene product. 361 2


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