Sex determination in the social amoeba Dictyostelium discoideum

The genetics of sex determination remain mysterious in many organisms, including some that are otherwise well studied. Here we report the discovery and analysis of the mating-type locus of the model organism Dictyostelium discoideum. Three forms of a single genetic locus specify this species' three mating types: two versions of the locus are entirely different in sequence, and the third resembles a composite of the other two. Single, unrelated genes are sufficient to determine two of the mating types, whereas homologs of both these genes are required in the composite type. The key genes encode polypeptides that possess no recognizable similarity to established protein families. Sex determination in the social amoebae thus appears to use regulators that are unrelated to any others currently known.     

Immune-like phagocyte activity in the social amoeba

Social amoebae feed on bacteria in the soil but aggregate when starved to form a migrating slug. We describe a previously unknown cell type in the social amoeba, which appears to provide detoxification and immune-like functions and which we term sentinel (S) cells. S cells were observed to engulf bacteria and sequester toxins while circulating within the slug, eventually being sloughed off. A Toll/interleukin-1 receptor (TIR) domain protein, TirA, was also required for some S cell functions and for vegetative amoebae to feed on live bacteria. This apparent innate immune function in social amoebae, and the use of TirA for bacterial feeding, suggest an ancient cellular foraging mechanism that may have been adapted to defense functions well before the diversification of the animals.     

A chemoattractant receptor controls development in Dictyostelium discoideum

During the early stages of its developmental program, Dictyostelium discoideum expresses cell surface cyclic adenosine monophosphate (cyclic AMP) receptors. It has been suggested that these receptors coordinate the aggregation of individual cells into a multicellular organism and regulate the expression of a large number of developmentally regulated genes. The complementary DNA (cDNA) for the cyclic AMP receptor has now been cloned from lambda gt-11 libraries by screening with specific antiserum. The 2-kilobase messenger RNA (mRNA) that encodes the receptor is undetectable in growing cells, rises to a maximum at 3 to 4 hours of development, and then declines. In vitro transcribed complementary RNA, when hybridized to cellular mRNA, specifically arrests in vitro translation of the receptor polypeptide. When the cDNA is expressed in Dictyostelium cells, the undifferentiated cells specifically bind cyclic AMP. Cell lines transformed with a vector that expresses complementary mRNA (antisense) do not express the cyclic AMP receptor protein. These cells fail to enter the aggregation stage of development during starvation, whereas control and wild-type cells aggregate and complete the developmental program within 24 hours. The phenotype of the antisense transformants suggests that the cyclic AMP receptor is essential for development. The deduced amino acid sequence of the receptor reveals a high percentage of hydrophobic residues grouped in seven domains, similar to the rhodopsins and other receptors believed to interact with G proteins. It shares amino acid sequence identity and is immunologically cross-reactive with bovine rhodopsin. A model is proposed in which the cyclic AMP receptor crosses the bilayer seven times with a serine-rich cytoplasmic carboxyl terminus, the proposed site of ligand-induced receptor phosphorylation.     

Trehalase from Dictyostelium discoideum: purification and properties

The hydrolytic enzyme, trehalase, was isolated and purified approximately 90-fold from the cellular slime mold Dictyostelium discoideum. The purified trehalase has an optimal temperature of 45 degrees C and shows maximum activity at pH 5.5 in citrate buffer. Its Michaelis constant is 1.2x 10(-3)M.     

Phosphodiesterase in Dictyostelium discoideum and the chemotactic response to cyclic adenosine monophosphate

A phosphodiesterase with a low Michaelis constant for cyclic adenosine monophosphate was found in the membrane fraction of the cellular slime mold. This activity was highest during the aggregation stage. Enzyme with similar properties was also secreted by the cells. Dithiothreitol inhibited both enzymes and potentiated the cellular response to cyclic adenosine monophosphate.     

Dictyostelium discoideum: a model system for cell-cell interactions in development

The cellular slime mold Dictyostelium discoideum undergoes a transition from single-celled amoebae to a multicellular organism as a natural part of its life cycle. A method of cell-cell signaling that controls chemotaxis, morphogenesis, and gene expression has developed in this organism, and a detailed understanding of this signaling system provides clues to mechanisms of intercellular communication in the development of metazoans.     

Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum

The role of myosin in the contraction of striated muscle cells is well known, but its importance in nonmuscle cells is not yet clear. The function of myosin in Dictyostelium discoideum has been investigated by isolating cells which specifically lack myosin heavy chain (MHC A) protein. Cells were transformed with a vector encoding RNA complementary to mhcA messenger RNA (antisense RNA). Stable transformants have a dramatic reduction in the amount of MHC A protein, grow slowly, and generate giant multinucleated progeny, indicating an impairment in cytokinesis. Surprisingly, the cells adhere to surfaces, extend pseudopods and are capable of ameboid locomotion. The developmental sequence that is initiated by starving cells is severely impaired by the lack of myosin. The cells are unable to form multicellular aggregates normally and do not undergo subsequent morphogenesis. By changing the food source from liquid medium to bacteria, expression of the endogenous mhcA messenger RNA can be increased relative to expression of antisense RNA. When grown in this way, the transformed cells accumulate MHC A protein, remain mononucleate, and proceed through development normally.     

Cyclic nucleotide phosphodiesterase in Dictyostelium discoideum: interconversion of two enzyme forms

An extracellular cyclic nucleotide phosphodiesterase was isolated from either growing cultures or aggregating amoebas of Dictyostelium discoideum. The enzyme is released in a form with a low Michaelis constant (15 micromolar) and spontaneously undergoes a slow conversion to a less active form with a high Michaelis constant (2 mnillimolar). Inactivation was prevented or reversed by use of Cleland's reagent, dithiothreitol. The two enzyme forms may be part of a mechanism for control of concentration of cyclic adenosine monophosphate.     

Expression and characterization of a functional myosin head fragment in Dictyostelium discoideum

The isolated head fragment of myosin is a motor protein that is able to use energy liberated from the hydrolysis of adenosine triphosphate to cause sliding movement of actin filaments. Expression of a myosin fragment nearly equivalent to the amino-terminal globular head domain, generally referred to as subfragment 1, has been achieved by transforming the eukaryotic organism Dictyostelium discoideum with a plasmid that carries a 2.6-kilobase fragment of the cloned Dictyostelium myosin heavy chain gene under the control of the Dictyostelium actin-15 promoter. The recombinant fragment of the myosin heavy chain was purified 2400-fold from one of the resulting cell lines and was found to be functional by the following criteria: the myosin head fragment copurified with the essential and regulatory myosin light chains, decorated actin filaments, and displayed actin-activated adenosine triphosphatase activity. In addition, motility assays in vitro showed that the recombinant myosin fragment is capable of supporting sliding movement of actin filaments.     

Transfer RNA genes: landmarks for integration of mobile genetic elements in Dictyostelium discoideum

In prokaryotes and eukaryotes mobile genetic elements frequently disrupt the highly conservative structures of chromosomes, which are responsible for storage of genetic information. The factors determining the site for integration of such elements are still unknown. Transfer RNA (tRNA) genes are associated in a highly significant manner with different putative mobile genetic elements in the cellular slime mold Dictyostelium discoideum. These results suggest that tRNA genes in D. discoideum, and probably tRNA genes generally in lower eukaryotes, may function as genomic landmarks for the integration of different transposable elements in a strictly position-specific manner.     

Cyclic 3',5'-adenosine monophosphate phosphodiesterase produced by the slime mold Dictyostelium discoideum

An enzyme that hydrolyzes cyclic ',5'-adenosine monophosphate to 5'-adenosine monophosphate was found in the culture medium of the cellular slime mold Dictyostelium discoidum. The enzymatic activity shows a pH optimum of 7.5, and magnesium is required for maximum activity. The enzyme is not inhibited by caffeine. It has a Michaelis-Menten constant of 2 x 10(-3)M and its molecular weight is around 300,000.     

Control of Aggregation in Dictyostelium discoideum by an External Periodic Pulse of Cyclic Adenosine Monophosphate

We have induced and controlled normal aggregation of Dictyostelium discoideum amoebas by electrophoretic release of pulses of cyclic adenosine monophosphate from a microelectrode. This has yielded information about the sequence of development of aggregation competences during interphase. We believe that modifications of the technique will have wide application in investigations of other developing systems.     

Adenosine 3',5'-monophosphate waves in Dictyostelium discoideum: a demonstration by isotope dilution--fluorography

The distribution of adenosine 3',5'-monophosphate (cyclic AMP) in fields of aggregating amoebae of Dictyostelium discoideum was examined by a novel isotope dilution-fluorographic technique. Cellular cyclic AMP was visualized by its competition with exogenous 3H-labeled cyclic AMP for high-affinity binding sites on protein kinase immobilized on a Millipore filter used to blot the monolayer. The cyclic AMP was distributed in spiral or concentric circular wave patterns which centered on the foci of the aggregations. These patterns were correlated with those of cell shape change that propagate through the monolayers: cells in regions of high concentrations of cyclic AMP were elongated (presumably moving up a cyclic AMP gradient), whereas those in regions of low cyclic AMP concentrations were randomly directed. The highest cyclic AMP concentrations were about 10(-6)M. The widths of the regions of elevated cyclic AMP were about 0.3 to 1 millimeter which, assuming a wave velocity of 300 micrometers per minute, suggests that a cell signals for about 1 to 3 minutes. These observations support the hypothesis that the aggregation process in Dictyostelium is mediated by the periodic relay of cyclic AMP signals and suggest a simple scheme for the dynamics of the aggregation process.     

DNA分子标记技术在作物单基因抗逆性育种中的应用

20世纪 80年代以后 ,分子生物学技术得到了快速的发展 ,在有关生命科学的各个领域都得到了广泛地应用 ,改变了许多生命科学传统的研究方法。在作物抗病育种过程中 ,一些隐性的及受环境因子影响较大等不易通过田间抗性表现型筛选的抗病基因 ,如抗病毒基因、抗霜霉病基因等大都是隐性遗传的 ;植物线虫病的抗性也难以通过大规模的田间接种与表现型观察来评价。利用DNA分子标记技术 ,即通过选择与抗病基因紧密连锁的特定序列的DNA分子标记 ,是提高上述类型抗病基因的抗原筛选鉴定、杂交分离后代汰选工作的效率与准确性的有效途径。DNA分子标…     

基于中国鉴别品种及单基因品系的湖北省稻瘟病菌的致病型分布

基于中国鉴别品种和单基因品系,采用离体点液接种方法对湖北省水稻主栽区稻瘟病菌的生理小种进行了鉴定。根据监测结果,发现2008～2009年这两年中生理小种ZA群出现频率略微下降,ZB、ZC群的出现频率则在缓慢上升,总体上ZA群、ZB群和ZC群的出现频率相差不大。2009年的100个菌株基于单基因品系出现毒性类型分散,毒性类型的丰富度为3.46。     

Membrane recycling: vesiculation of the amoeba contractile vacuole at systole

Ultrastructural data on the protozoan Amoeba proteus support a model of membrane recycling. At systole the amoeba contractile vacuole fuses with the cell surface and expels its contents. Observations by electron microscopy indicate that, as the vacuole empties, its bounding membrane transforms into tiny (35 nanometers in diameter) vesicles, identical to the vesicles that segregate fluid and contribute to the diastolic vacuole.     

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