bluuebunny/crossref_metadata_2025 huggingface dataset:
The cyclic nucleotide phosphodiesterase of Dictyostelium discoideum: The structure of the gene and its regulation and role in development
Authors: Podgorski Gregory J. , Faure Michel , Franke Jakob , Kessin Richard H.
Published: May 2005

The cyclic nucleotide phosphodiesterase (phosphodiesterase) of Dictyostelium discoideum plays an essential role in development by hydrolyzing the cAMP used as a chemoattractant by aggregating cells. We have studied the biochemistry of the phosphodiesterase and a functionally related protein, the phosphodiesterase inhibitor protein, and have cloned the cognate genes. A 1.8‐kb and a 2.2‐kb mRNA are transcribed from the singlephosphodiesterase gene. The 2.2‐kb mRNA comprises the majority of the phosphodiesterase mRNA found in differentiating cells and is transcribed only during development from a promoter at least 2.5 kb upstream of the translational start site. The 1.8‐kb phosphodiesterase mRNA is detected at all stages of growth and development, is present at lower levels than the developmentally induced mRNA, and is transcribed from a site proximal to the protein‐coding region. The phosphodiesterase gene contains a minimum of three exons, and a 2.3‐kb intron, the longest yet reported for this organism. We have shown that the pds A. gene and fourfgd genes affect, the accumulation of the phosphodiesterase mRNAs, and we believe that these loci represent a significant portion of the genes regulating expression of the phosphodiesterase. The phosphodiesterase gene was introduced into cells by transformation and used as a tool to explore the effects of cAMP on the terminal stages of development. In cells expressing high levels of phosphodiesterase activity, final morphogenesis cannot be completed, and differentiated spore and stalk cells do not form. We interpret these results to support the hypothesis that cAMP plays an essential role in organizing cell movements in late development as well as in controlling the aggregation of cells in the initial phase of the developmental program.
Contact alters cAMP metabolism in aggregation‐competent Dictyostelium amoebae
Authors: Fontana Donna R. , Price Pamela L.
Published: May 2005

cAMP and cell‐cell contact are involved in the coordination of differentiation and morphogenesis in Dictyostelium discoideum. The experiments described in this paper establish a relationship between cAMP and cell‐cell contact. Contact between Enterobacter aerogenes and aggregation‐competent Dictyostelium amoebae and contact between Dictyostelium amoebae themselves results in the transient secretion of cAMP and an alteration in the amount of cAMP secreted in response to subsequent stimulation by cAMP, i.e., an alteration in magnitude of a cAMP relay response. The subsequent cAMP relay response can be enhanced or diminished depending upon the number of contacts formed and the concentration of cAMP present at the time of contact. Latex beads are capable of evoking cAMP secretion. However, the bead/amoebal contact is unable to alter the magnitude of a subsequent response to cAMP. This suggests that a nonspecific interaction via cell‐cell contactelicits transient cAMP secretion in aggregation‐competent Dictyostelium amoebae.

The two responses to cell‐cell contact are distinct from each other and distinct from the cAMP relay response. (1) The dose‐response curves for the responses to Enterobacter contact are clearly different. (2) Contact with latex beads can elicit cAMP secretion but not alter the magnitude of a subsequent cAMP relay response. (3) The temperature dependences of the contact‐induced responses and the cAMP relay response show that only the contact‐induced cAMP secretion is inhibited at 12 and 15°C, while only the cAMP relay response is inhibited at 28°C.

A 4‐second application of cAMP at the time that contact is initiated enhances both contact‐induced responses. Whether the relationship between these two developmental regulators is important for the regulation of Dictyostelium development has yet to be established.
The effect of caffeine, adenosine, and buffer ionic composition on the induction of cell‐surface cAMP binding during starvation of Dictyostelium discoideum
Authors: Drummond Iain A. S. , Chisholm Rex L.
Published: May 2005

We have analyzed the effects of the cAMP relay inhibitor, caffeine, and the receptor antagonist, adenosine, on the regulation of the cell‐surface cAMP receptor in suspensionstarved Dictyostelium discoideum cells by measuring ammonium sulfate‐stabilized binding of [3‐H]cAMP to intact cells. When cells were starved in fast (230 r.p.m.) shaken suspension in 10 mM Na+/5 mM K+ phosphate buffer, pH 6.5, plus 1 mM CaCl2 and 2.5 mM MgCl2, and assayed for specific cAMP binding, receptor accumulation peaked at approximately 6 hours, reaching a maximum of 1.5 pmol cAMP bound/107 cells (saturation binding). Neither caffeine nor adenosine inhibited the accumulation of cAMP receptors. Similar results were obtained in caffeine‐treated, slow shaken (90 r.p.m.) suspension cultures. These results suggest that starvation alone is sufficient stimulus to induce the cAMP receptor. We have also tested the effects of different buffer ionic compositions on the accumulation of cAMP receptors. Elevation of the monovalent ion concentration to 30‐40 mM was found to significantly inhibit the induction of cAMP receptors.
Characterization of genes that are developmentally regulated during Dictyostelium discoideum spore germination
Authors: Ennis Herbert L. , Giorda Roberto , Ohmachi Tetsuo , Shaw David R.
Published: May 2005

Similar to other stages in Dictyostelium development, spore germination is a particularly suitable model for studying the regulation of gene expression, because developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. Spores are constitutively dormant and must be activated to germinate. Under the proper environmental conditions, spores germinate in a highly synchronous manner to give rise to individual amoebae that can then enter the vegetative growth phase. Protein synthesis is developmentally regulated during this process. Because protein synthesis is transcriptionally controlled during spore germination, the respective genes must be developmentally transcribed, and these can be isolated and analyzed. Three cDNA clones specific for mRNA developmentally regulated during spore germination have been characterized and used as probes to study mRNA accumulation and decay during spore germination. Because we are interested in defining the sequences of developmentally regulated genes that may relate to their regulation of transcription, we have sequenced the cDNAs and have isolated and sequenced their respective genomic clones. The sequences of the three gene families, their genomic organization, and their special structural features are described.
Analysis of the prestarvation response in growing cells of Dictyostelium discoideum
Authors: Clarke Margaret , Yang Jing , Kayman Samuel C.
Published: May 2005

We have previously shown that growing cells of Dictyostelium discoideum (strains NC4 and AX3) produce a soluble substance that accumulates in the medium in proportion to cell density; this substance regulates the production of certain proteins previously thought to be induced by starvation [Clarke et al., 1987]. We suggest the name PSF (prestarvation factor) for this substance. During growth, Dictyostelium cells monitor the relative concentrations of PSF and food bacteria. When PSF reaches a sufficiently high level relative to the concentration of bacteria, synthesis of PSF‐regulated proteins is induced. We propose the name prestarvation response for this induction, which takes place in exponentially growing cells several generations before the food bacteria are depleted. We have explored the mechanism by which the food bacteria inhibit the response of Dictyostelium cells to PSF. We find that the bacteria do not inactivate PSF or inhibit its production; instead, they affect the ability of NC4 cells to detect PSF, possibly by binding to the same cell surface receptor. In the absence of bacteria, as during axenic growth of AX3 cells, the prestarvation response occurs at much lower cell densities, probably accounting for the presence of certain developmentally regulated mRNAs and proteins in axenic cultures.
Deactivation of gene expression upon the onset of development in Dictyostelium discoideum
Authors: Singleton Charles K. , McPherson Clifton E. , Manning Suzanne S.
Published: May 2005

Several genes that are deactivated upon the initiation of development of Dictyostelium discoideum have been identified by differential screening of various cDNA libraries. These genes have in common a decrease in the steady‐state levels of their corresponding mRNAs 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. However, for about 5% of the genes (H genes), 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 trascription rate; normal rates of transcription for the H genes were dependent upon continued protein synthesis during vegetative growth and during development. Thus, two general regulatory classes exist for deactivation of gene expression upon initiation of development, one dependent and one independent of protein synthesis. Models concerning the control of expression of these two classes of genes are discussed here. Analysis of expression of these genes in mutant strains that are aggregation‐deficient has also been performed, and the results lead to subdivisions of the classes.
Control of early gene expression in Dictyostelium
Authors: Mann Sandra K. O. , Pinko Christopher , Firtel Richard A.
Published: May 2005

We have examined the expression of a cAMP pulse‐repressed and two cAMP pulse‐induced genes in response to cAMP and caffeine under a number of different physiological conditions, and in several classes of developmental mutants altered in cAMP‐mediated signal transduction pathways. The data presented help characterize the mutants with regard to early gene expression. Analysis of the data indicates that full induction of the pulse‐induced or repression of the pulse‐repressed genes requires cycles of activation and adaptation of the cAMP receptor but does not require a rise in intracellular cAMP. Comparison of the results obtained between different mutant classes suggests that repression and activation of the two classes of genes can be uncoupled, implying that different intracellular mechanisms control these processes. In addition, we examined the effects of caffeine and show that it can induce pulse‐induced mRNA accumulation in the absence of cAMP.
Transmembrane signal transduction regulates gene expression in Dictyostelium discoideum
Authors: Pavlovic Jovan , Haribabu Bodduluri , Dottin Robert P.
Published: May 2005

cAMP regulates gene expression in Dictyostelium discoideum through the cell surface receptor and is therefore a transmembrane signal transduction event. We have now begun to examine the signal transduction pathway that transmits the cAMP‐induced signal to the nucleus. The results presented here indicate that Ca2+ plays a crucial role. A comparison of the accumulation of UDPGP1 mRNA during development with the corresponding transcription rates revealed that this gene is regulated primarily at the level of transcription. To elucidate the factors involved in the regulation of the UDPGP1 gene we characterized its cis acting sequences. We constructed a series of deletions into the 5′ flanking region of the UDPGP1 gene and analyzed the expression of the mutated DNA in transformants. A sequence element essential for the expression of the UDPGP1 gene is located between ‐500 bp and ‐288 dp from the transcription start site. This promoter element appears to be a short G + C‐rich sequence positioned between ‐374 to ‐395 and coincides with a DNase I hypersensitive site.
Structural and functional characterization of genes encoding Dictyostelium prestalk and prespore cell‐specific proteins
Authors: Early Anne , McRobbie Stuart J. , Duffy Karen T. , Jermyn Keith A. , Tilly Rita , Ceccarelli Adriano , Williams Jeffrey G.
Published: May 2005

The nucleotide sequence of D19, a Dictyostelium gene that encodes a prespore‐specific mRNA sequence shows it to encode PsA, the cell surface protein detected by the MUD 1 monoclonal antibody. The predicted sequence of the protein reveals a largely hydrophobic C terminus, with chemical similarity to proteins known to be attached to the plasma membrane via a phosphatidylinositol link. The C‐terminal region has direct sequence homology to the contact sites A protein and to the phosphatidylinositol‐linked form of a chicken N‐CAM, suggesting that it might play a role in cell adhesion. Expression of the D19 gene is known to be induced by cAMP and repressed by adenosine. The accumulation of the D19 mRNA is also repressed by DIF, the putative stalk‐specific morphogen, and this effect is mediated at the transcriptional level. The pDd56 and pDd63 genes are induced by DIF, and they are specific markers of prestalk and stalk cells. They encode, respectively, ST310 and ST430, two proteins that were first identified by two‐dimensional gel electrophoresis. Both proteins are predominantly composed of a highly conserved, 24‐amino acid repeat. The two proteins are localized in the slime sheath of the migratory slug and in the stalk tube and stalk cell wall of the mature culminant, where they presumably function as structural components of the extracellular matrix. We have constructed marked derivatives of the pDd56, pDd63, and D19 genes, and these are correctly regulated after transformation into Dictyostelium cells. Thus we have determined the structure, and elucidated possible functions, for one prespore and two prestalk genes. These sequences should be of value, both as markers of the earliest events in cellular differentiation and in identifying the regulatory sequences controlling cell type‐specific gene expression.
Regulation of mRNA stability and the poly(A) problem in Dictyostelium discoideum
Authors: Manrow Richard E. , Shapiro Robert A. , Herrick David , Steel Laura F. , Blinder Dmitry , Jacobson Allan
Published: May 2005

This paper reviews our studies of three aspects of post‐transcriptional regulation in Dictyostelium discoideum: (1) the determinants of mRNA stability in vegetative amoebae; (2) the effects of disaggregation and cyclic AMP on the decay rates of cell‐type‐specific mRNAs in late developing cells; and (3) the cytoplasmic function of the 3′ poly(A) tracts present on most mRNAs. We find that: (1) mRNA stability in vegetative amoebae is not dependent on mRNA size, ribosome loading, or poly(A) tract length, but may be determined by specific 3′‐untranslated sequences within a given mRNA; (2) mRNA decay rates in late developing cells are heterogeneous, and cyclic AMP does not act directly to stabilize cell‐type‐specific mRNAs; and (3) poly(A) is most likely involved in the initiation of protein synthesis via an interaction with cytoplasmic poly(A)‐binding proteins.
Post‐transcriptional regulation of ribosomal protein gene expression during development in Dictyostelium discoideum
Authors: Steel Laura F. , Jacobson Allan
Published: May 2005

We have isolated recombinant plasmids that contain cDNA inserts complementary to mRNAs encoding six different r‐proteins of Dictyostelium discoideum. Southern and quantitative dot blot analyses have shown that each of the r‐protein genes represented in these plasmids is encoded by a single copy gene and that these genes are not tightly linked to each other. We have determined the relative amount of the six r‐protein mRNAs present in cells at intervals throughout development and find that for the first 9 hours of development, each of the mRNAs remains present at virtually the same level as in vegetatively growing cells. Between 9 and 11 hours of development, there is a rapid loss of these mRNAs to 15% or less of vegetative levels, and that low level remains, or slightly declines, through the late stages of development. We have shown that two post‐transcriptional events contribute to the developmental regulation of the expression of the r‐protein genes. The first involves a specific block to translational initiation that is not the result of inactivation of these mRNAs by decapping or deadenylation. The second is a change in the stability of these mRNAs during early development. In order to begin to analyze the role of specific sequences that may act as targets or signals in these events, we have cloned and sequenced a 1.9‐kb genomic DNA fragment that encodes one of the r‐proteins. We find that transcription of this gene begins in a pyrimidine‐rich region that is not preceded by a TATA box, the gene contains a single intron of 350 bp, and there are two alternative 3′ processing sites. In addition, the 5′‐untranslated region of the transcript contains an unusually high percentage of G and C residues relative to other Dictyostelium mRNAs.
DNA repair in Dictyostelium
Authors: Deering Reginald A.
Published: May 2005

Recent approaches to the study of DNA repair in Dictyostelium discoideum are reviewed. Thymidine auxotrophs facilitate the uptake of labeled thymidine into DNA during its replication and repair. The tmpA 600 mutation leads to a loss of thymidylate synthase activity, and tdrA600 results in increased transport of thymidine into the cell. In the HPS401 double mutant (tmpA600tdrA600), thymidine is taken up uniformly into the nuclear and mitochondrial DNAs at levels up to 50‐fold that in the wild type. tmpA maps on linkage group III. tdrA is on IV or VI, which cosegregate in strains containing this mutation. Alkaline sucrose gradients of nuclei from HPS401 pulsed for 15 min with [3H]thymidine in axenic medium show that the initially labeled single‐strand DNA is about 7 × 106 daltons, which may be the size of the replicon. This nascent DNA matures in about 45 minutes to 2 × 108 daltons. Ultraviolet light (254 nm) decreases the size of the nascent DNA and delays its maturation. In addition to studies of DNA repair utilizing repairproficient and ‐deficient mutants of thymidine auxotrophs, we are currently using two approaches for cloning genes involved in repair: (1) genes are sought that can functionally complement repair defects in Saccharomyces cerevisiae following transformation with a D. discoideum DNA library in YEp 24(URA); 4‐NQO is used for the selection of RAD transformants; and (2) we have characterized and purified to near‐homogeneity two repair enzymes from D. discoideum–uracil‐DNA glycosylase and AP‐endonuclease. An Nterminal sequence has been determined for the glycosylase, and a synthetic oligonucleotide probe derived from this sequence will be used to screen for this gene. A similar approach is in progress for the AP‐endonuclease.
Electron microscopic localization of myosin II and ABP‐120 in the cortical actin matrix of Dictyostelium amoebae using IgG‐gold conjugates
Authors: Ogihara S. , Carboni J. , Condeelis J.
Published: May 2005

To narrow the field of possible functions of an actin‐binding protein (ABP‐120) and myosin II, we have used high resolution immunocytochemistry with IgG‐colloidal gold conjugates to identify the types of actin containing structures with which these proteins are associated in the isolated cell cortex. Staining for myosin II and ABP‐120 is associated with distinct regions of the actin cytoskeleton in isolated cortices. Myosin II is localized to lateral arrays of filaments, where it is clustered and has a density that is unrelated to distance from the plasma membrane. Staining for myosin II is associated also with unidentified cytoplasmic vesicles. However, staining for ABP‐120 is concentrated in dense networks of branched microfilaments that are adjacent to the plasma membrane or in surface projections (residual pseudopods and lamellopods). These results are consistent with a role for ABP‐120 in the formation of filament networks in vivo and further suggest that networks of branched microfilaments are unlikely to participate in motility that is mediated by myosin II.
Movement of the Dictyostelium discoideum slug: Models, musings, and images
Authors: Breen Edmond J. , Williams Keith L.
Published: May 2005

The last 5 years have resulted in many advances in knowledge of the cytoskeleton and motility of individual cells. Here the problem of multicellular movement is addressed. The Dictyostelium discoideum slug is examined, and models for how approximately 100,000 cells become coordinated to move are briefly reviewed. Experiments that contributed to model building as well as those used to test models are considered. Four levels of experimentation are considered: (1) the extracellular matrix (ECM) is examined as a component of the system; (2) information obtained by examining the organisation of slug cells through sectioning is presented; (3) time, the 4th dimension, is considered, and approaches to studying the dynamics of cell interactions from the point of view of movement are outlined, and (4) cell adhesion molecules are addressed.
Cell‐cell adhesion in Dictyostelium discoideum
Authors: Loomis William F.
Published: May 2005

Three separate mechanisms of cell‐cell adhesion have been shown to appear at different stages of development in Dictyostelium discoideum. During the first few hours of development, the cells synthesize and accumulate a glycoprotein of 24,000 daltons (gp24) that is positioned in the membrane. The time of appearance of gp24 correlates exactly with the time of appearance of cell‐cell adhesion in two strains in which temporal control varies by several hours. Antibodies specific to gp24 are able to block cell‐cell adhesion during the first few hours of development but not during later development. By 8 hr of development, another glycoprotein, gp80, that is not recognized by antibodies to gp24 accumulates on the surface of cells. This membrane protein mediates an independent adhesion mechanism during the aggregation stage that is resistant to 10 mM EDTA. Antibodies specific to gp80 can block EDTA‐resistant adhesion during this stage. During subsequent development, gp80 is removed from the cell surface and replaced by another adhesion mechanism that is insensitive to antibodies to either gp24 or gp80.

A λgtll expression vector carrying a Dictyostelium cDNA insert was isolated that directs the synthesis of a fusion protein recognized by antibodies specific to gp24. This cDNA was used to probe a genomic library. A clone carrying a 1.4‐kb insert of genomic DNA was recognized by the cDNA and shown to hybridize to a 0.7‐kb mRNA that accumulates early in development. This unusually small RNA could code for the small protein, gp24. Southern analysis of restriction fragments generated by various enzymes on Dictyostelium DNA with both the cDNA and genomic clones indicated the presence of two tandem copies of the gene. This may account for the failure to recover mutations resulting in the lack of gp24.

Mutations have been recovered that result in the lack of accumulation of gp80, and cells carrying these mutations have been shown to be missing the second adhesion mechanism. These mutant strains are able to complete development because the other adhesion mechanisms are not impaired. Sequential addition of adhesion mechanisms provides a means for the formation of multicellular organisms from previously solitary cells.
Biosynthesis of 117 antigen: A cell cohesion molecule in Dictyostelium discoideum
Authors: Sadeghi Homayoun , Silva Aline Da , Klein Claudette
Published: May 2005

117 antigen is involved in the process of intercellular cohesiion in Dictyostelium discoideum [Brodie et al., 1983]. The antigen, a 69‐and 72‐kDa doublet, was found to arise from a 60‐and 62‐kDa precursor. The mature antigen contains N‐linked oligosaccharides that are sulfated and fucosylated [Sadeghi et al., 1987]. These oligosaccharide chains are resistant to endoglycosidase H digestion. 117 antigen also contains a post‐translationally added carbohydrate‐containing modification(s). Unlike the N‐linked oligosaccharide, this carbohydrate moiety is sensitive to periodate oxidation. 117 antigen is developmentally regulated, and the changes in rate of 117 antigen synthesis reflect changes in the cellular levels of its mRNA. 117 mRNA accumulates in starving cells and reaches its maximum when cells become aggregation competent. The mRNA levels then decline, and by the time the slug structure is formed, no 117 mRNA is present. 117 mRNA reaccumulates for a brief period during early culmination and then returns to an undetectable level.
Cell behavior during formation of prestalk/prespore pattern in submerged agglomerates of Dictyostelium discoideum
Authors: Takeuchi Ikuo , Kakutani Tetsuji , Tasaka Masao
Published: May 2005

When cells dissociated from Dictyostelium discoideum slugs were cultured in roller tubes, they formed agglomerates in which prestalk cells were initially dispersed but soon sorted out to the center and then moved to the edge to reconstitute the prestalk/prespore pattern. To examine the mechanism of sorting out, individual prestalk cells were traced by a videotape recorder. The radial component of the rate of movement toward the center of the presumptive prestalk region was calculated. Prestalk cells did not move randomly, but rather directionally toward the center. Thei movement was pulsatile, with a period of ca. 15 min, and accompanied by occasional formation of cell streams, thus resembling the movement observable during cell aggregation. These results favor the idea that prestalk cells sort out to the prestalk region due to differential chemotaxis rather than differential adhesiveness. After formation of the prestalk/prespore pattern, the prestalk region rotated along the circumference of the agglomerates. This appears comparable to migration of slugs on the substratum, the rate of rotation being similar to that of slug migration.

To examine the processes of pattern formation during development. washed vegetative cells were cultured in roller tubes. Prespore cells identified by antispore immunoglobulin initially appeared randomly within the agglomerates, but then nonprespore cells accumulated in the center and finally moved to the edge to establish the prestalk/prespore pattern, the processes being similar to those of pattern reconstruction with differentiated prestalk and prespore cells.
A mutant strain of Polysphondylium with defects in many genes
Authors: Francis David , Shaffer Arthur
Published: May 2005

PN6024 is a mutant strain of P. pallidum which appeared on selection for resistance to MDMP, an inhibitor of translation. It was found to be mutant in four other traits, being resistant to tubercidin, incapable of growth at 31.5°C, abnormal in development, and slow growing at 25°C. Genetic crosses using the macrocyst cycle showed that these five traits are controlled by five unlinked genes. The hypothesis is that movement of a transposon to multiple new locations caused these mutations. A difference in restriction fragment pattern between PN6024 and its parent PN600 support the hypothesis. Attempts were made to find conditions generating other strains like PN6024. Selection for growth in the presence of tubercidin yielded clones which resemble PN6024 in being developmentally abnormal as well as tubercidin resistant. Tubercidin treatment also increased the frequency of clones resistant to canavanine. It is suggested that tubercidin is mutagenic because it causes movement of the putative transposon, not because it generates point mutations. Growth under conditions of stress (at 31.5°C, at 8°C, in the presence of 2% ethanol) had at most an erratic effect in generating strains like PN6024.

Three substrains appeared spontaneously in cultures of PN6024. These differed in developmental characteristics from each other and from the parent strain. It is suggested that they carry mutations in genes which control the choices between growth and aggregation, and between aggregation and encystment.
Ammonia can stimulate or inhibit aggregate density in Dictyostelium mucoroides: A quantitative test of the hypothesis that ammonia is the aggregation‐suppressing gas
Authors: Feit Ira N.
Published: May 2005

Ammonia, at moderate concentrations, stimulates aggregate density of Dictyostelium mucoroides. The range of stimulatory concentrations includes ammonia concentrations established by populations of amebae. At higher concentrations, ammonia inhibits aggregate density.

A quantitative test of the hypothesis that ammonia is the aggregation‐suppressing gas has been carried out. The concentration of ammonia established over defined populations of amebae is one or two orders of magnitude lower than the concentration of ammonia required to exert the same degree of inhibitory effect as the populations of amebae exert.

An additional difference between ammonia and the aggregation‐suppressing gas is the fact that increasing concentrations of the aggregation‐suppressing gas cause progressively larger aggregation streams, while increasing concentrations of ammonia have no such effect.

The stimulatory effect of ammonia at concentrations established by ameba populations indicates that ammonia must be included in the variables affecting the aggregation process and that this ammonia effect must be taken into account in any quantitative modelling of the aggregation process.
Purification, characterization, and partial structure of D factor from Polysphondylium violaceum
Authors: Hanna Michael H. , Fatone Michael , Newth‐Clark Cynthia , Salerno John , Clemans Steven
Published: May 2005

The A component of D factor (DfA) was overproduced during development of wild type Polyspondylium violaceum strain China after starvation in liquid medium. Crude DfA excreted by strain China was partially purified by ultrafiltration using Amicon YM10 and YM2 filters with DfA extracted from the filtrate by absorption onto a preparative grade C‐18 resin. The concentrated material was further purified on a C‐18 analytical column using both acetonitrile:water and methanol: water gradients. This highly purified fraction was a single component with a final specific activity of greater than 106 units per mg dry weight. Purified DfA is red having a broad visible absorbance at 500 nm and a ultraviolet (uv) absorbance at 290–300 nm. The red chromophore is sensitive to pH and to oxidation‐reduction. 1H and 13C nmr studies with purified DfA indicate that it is a C11 compound with both polar and non‐polar regions. The non‐polar region has been identified as a hexanone and is the same as the side chain of DIF from Dictyostelium discoideum. Purified DfA has been used in studies with the D factor non‐producing mutant, tsg‐119 cyc‐1 aggA586 (A586), to show that neither production of glorin nor chemotactic sensitivity to glorin are affected by D factor. However, founder cells develop in A586 mutant populations only after addition of D factor. These data suggest that DfA may be necessary for induction of aggregate formation by aggregation‐competent amoebae.
Cell size in Dictyostelium
Authors: Waddell David R.
Published: May 2005

Cellular slime mold amoebae have become a model system for the study of cell motility and the cytoskeleton. A basic problem which all cells face that involves the cytoskeleton is how to control their size. The varied ways in which cellular slime mold amoebae change their cell size‐by changing the size at which division occurs, by cell fusion, and by control over cytokinesis‐are reviewed. A model is presented which attempts to explain how the mechanisms affected in certain cytokinesis mutants in Dictyostelium discideum known as phg mutants could be involved in control of cell size in the predatory slime mold Dictyostelium caveatum.
Changes in protein synthetic activity in early Drosophila embryos mutat for the segmentation gene Krüppel
Authors: Bedian Vahe , Summers Michael C. , Kauffman Stuart A.
Published: May 2005

We have identified early embryo proteins related to the segmentation gene Krüppel by [35S]methionine pulse labelling and two‐dimensional gel electrophoresis. Protein synthesis differences shared by homozygous embryos of two Krüppel alleles when compared to heterozygous and wild‐type embryos are reported. The study was extended to syncytial blastoderm stages by pulse labelling and gel analysis of single embryos, using Krüppel specific proteins from gastrula stages as molecular markers for identifying homozygous Krüppel embryos. Localized expression of interesting proteins was examined in embryo fragments. The earliest differences detected at nuclear migration stages showed unregulated synthesis in mutant embryos of two proteins that have stage specific synthesis in normal embryos. At the cellular blastoderm stage one protein was not synthesized and two proteins showed apparent shifts in isoelectric point in mutant embryos. Differences observed in older embryos included additional proteins with shifted isoelectric points and a number of qualitative and quantitative changes in protein synthesis. Five of the proteins with altered rates of synthesis in mutant embryos showed localized synthesis in normal embryos. The early effects observed are consistent with the hypothesis that the Krüppel product can be a negative or positive regulator of expression of other loci, while blastoderm and gastrula stage shifts in isoelectric point indicate that a secondary effect of Krüppel function may involve post‐translational modification of proteins.
Pretranslational control of the levels of glyoxysomal protein gene expression by the embryonic axis in maize
Authors: Skadsen Ronald W. , Scandalios John G.
Published: May 2005

Previous studies showed that the expression of catalase‐2 (CAT‐2) and other glyoxysomal proteins is independently controlled in the scutella of intact maize seedlings. In this study, removal of the embryonic axis prior to seed imbibition dramatically decreased the amounts of all but two of the 19 immunologically detectable glyoxysomal proteins in the scutellum, including CAT‐2. The temporal expression profile of CAT‐2 was also altered. Removal of the axis after seeds were fully imbibed (24 hr) had little effect on the subsequent pattern of expression of CAT‐2. The effect of axis removal was specific for glyoxysomal enzymes and caused relatively little change in the population of stainable scutellar proteins. In vitro translation studies and nucleic acid hybridization with a gene‐specific cloned probe (for Cat2) revealed that the mRNA levels for glyoxysomal proteins were sharply lowered by axis removal. This study provides evidence that a signal may be released from the embryonic axis during imbibition, leading to the expression of a set of glyoxysomal enzymes by enhancing either the transcription of their genes or transcript stability.
Effects of heat shock on protein processing and turnover in developing Drosophila wings
Authors: Petersen Nancy S. , Young Patricia
Published: May 2005

Developmental defects called phenocopies can be induced by heating Drosophila melanogaster pupae at specific developmental stages. The induction of the defects is thought to be a result of interference with gene expression at some level (Petersen and Mitchell, Dev Biol 1987; 121:335‐341, 1987). Here we look at protein turnover in developing 52‐hour wings and at the effect of heat on the proteolytic processing of three proteins that normally turn over rapidly. The effect of the heat treatment itself on the turnover of each protein is different. However, all of the proteins appear to be stabilized at 25°C during recovery from severe heat shocks.
Sex‐, tissue‐, and stage‐specific expression of a vitelline membrane protein gene from region 32 of the second chromosome of Drosophila melanogaster
Authors: Gigliotti S. , Graziani F. , De Ponti L. , Rafti F. , Manzi A. , Lavorgna G. , Gargiulo G. , Malva C.
Published: May 2005

This study isolated cDNA clones from egg‐chamber and adult female Drosophila cDNA libraries using as probe a DNA fragment from a 200‐kb “chromosome walk” in region 32E of the second chromosome of D. melanogaster. The present authors believe that these clones correspond to a new vitelline membrane protein (VMP) gene because (1) cDNA clones in Northern blots identify a transcript expressed in a tissue‐ and stage‐specific manner: stage 10 egg‐chambers; (2) the sequence of cDNAs and of the genomic subclone shows homology with the other VMP genes that have been identified to date; (3) the amino acid composition of the translational product has the high content of proline and alanine characteristic of VMPs. Two aspects emerging from this study are worth stressing: (1) the presence of a hydrophobic domain that is highly conserved in all the VMP genes; and (2) the particularly narrow period of expression of the isolated gene, which could be related to the mechanism of vitelline membrane assembly.
The role of polyfusomes in generating branched chains of cystocytes during Drosophila oogenesis
Authors: Storto Patrick D. , King Robert C.
Published: May 2005

Three‐dimensional models were constructed utilizing the information gained from electron micrographs of serial sections of two clones of cystocytes undergoing their terminal divisions. In each clone a polyfusome connected all eight cystocytes together. Each of the spindles was oriented so that one pole touched the polyfusomes, while the other pointed away from it. This positioning of spindles ensures that one cell of each dividing pair retains all previously formed canals, while the other receives none. The two cells that eventually come to contain the maximum number of canals and fusomal material are the ones that differentiate as pro‐oocytes, while the others become nurse cells. The orientation of each spindle suggests that the polyfusome formed at one division determines the placement of the cytoskeletal fibers that anchor the spindles formed at the next division. There is a centripetal gathering together of new canals following each cycle of cystocyte division, which is thought to result from the subsequent contraction of the polyfusomal system. Females homozygous for the otu1 mutation are characterized by ovarian tumors, which result when germarial cystocytes undergo supernumerary divisions and fail to differentiate into either nurse cells or oocytes. An analysis of electron micrographs taken of serially sectioned, mutant germaria showed that most germ cells were single or belonged to clusters of two or three interconnected cells. Therefore otu1 cystocytes are unable to undergo a sustained series of arrested cleavages. These cystocytes contain fusomal material that shows ultrastructural differences from normal polyfusomes. We conclude: (1) that a normal polyfusomal system is a necessary prerequisite for the production of a branched chain of cystocytes and for their subsequent differentiation into pro‐oocytes and nurse cells; and (2) that a product encoded by the otu+ gene is essential for the construction of a functional polyfusome.
Regulated expression of genes encoding soybean β‐conglycinins in transgenic plants
Authors: Chen Zhang‐Liang , Naito Satoshi , Nakamura Ikuo , Beachy Roger N.
Published: May 2005

β‐conglycinin, an abundant storage protein in soybean seeds, is comprised of three subunits: α', α, and β. Expression of genes encoding these subunits is tissue‐specific and temporally regulated in soybean plants. Both the α'‐and β‐subunit genes have been isolated and fully or partially characterized by DNA sequence analysis. Each has been transferred to transgenic plants and was found to be expressed in petunia and tobacco plants in a regulated manner as in soybean plants. Both the α'‐ and β‐subunit proteins were accumulated only in transgenic seeds during mid‐to‐late stages of seed development and assembled into multimeric forms with sedimentation coefficients of 7S and 9S. The α'‐ and β‐subunit genes were also introduced on the same plasmid into transgenic plants. When the promoters of the two genes were proximal to each other, there was an increase in the expression of the β‐subunit gene, suggesting that there is a strong cis‐acting sequence in the α'‐gene (promoter) that can affect expression of the α‐subunit gene.

A series of deletion mutants was constructed in the α'‐gene promoter, and each was introduced into petunia plants. The DNA sequence approximately 250 bp upstream of the transcriptional initiation site contains sufficient regulatory information for expression of the α'‐subunit gene. The sequence about 200 bp upstream contains four 6‐bp repeats A (AGCCCA) that may play an important role in regulating expression of the α'‐gene. The function of this DNA sequence (‐78 to ‐257) was tested in a chimeric gene construct containing the CaMV 35S promoter, the CAT coding sequence, and the 3′ noncoding region of the α'‐gene. A dramatic enhancement of the CAT gene expression (at least 40‐fold) resulted when this DNA sequence was inserted into the 35S promoter at a position 90 bp upstream of the transcription start site, regardless of the orientation of the insert. There was a two‐ to threefold enhancement when the element was placed 3′ to the CAT coding region and no enhancement when placed downstream of the 3′ noncoding region. The enhancement was observed only in seeds during mid‐to‐late stages of seed development. This indicates that the 170‐bp DNA sequence can function as a strong cis‐acting element that controls seed‐specific and temporally regulated gene expression. This element is defined as a seed‐specific and developmentally regulated element (SDRE).
Molecular genetics of transformer, a genetic switch controlling sexual differentiation in Drosophila
Authors: Belote John M. , McKeown Michael , Boggs Russell T. , Ohkawa Risa , Sosnowski Barbara A.
Published: May 2005

The transformer gene is one of a set of regulatory genes that form the hierarchy controlling all aspects of somatic sexual differentiation in Drosophila melanogaster. The gene transformer occupies an intermediate position in this hierarchy. Analysis of this gene has allowed us to determine the mechanism by which it is regulated in a sex‐specific manner and to examine the way in which the regulatory hierarchy is organized. The female‐specific expression of the tra gene, previously inferred from genetic observations, is bused on sex‐specific alternative splicing of tra pre‐mRNA and is not the result of sex‐specific transcriptional activation. The female‐specific RNA produced by this alternative splicing is the functional mediator of tra activity. Multiple genetic, molecular, and transformation experiments show that female‐specific activation of genes or gene products occurs in the order Sex lethal > transformer > transformer‐2 > doublesex · intersex > female differentiation. The results do not distinguish the level at which transformer might regulate the downstream gene transformer‐2. Neither transformer nor any of the downstream genes feedback on, or participate in, alternative splicing of transformer RNA. The mechanism by which Sex lethal regulates transformer splicing appears to be a repression of the use of one of a pair of splice acceptor sites.
Effects of juvenile hormone on the ecdysone response of Drosophila Kc cells
Authors: Cherbas Lucy , Koehler M. Macy D. , Cherbas Peter
Published: May 2005

Drosophila Kc cells are ecdysone‐responsive: hormone treatment leads rapidly to increased synthesis of several ecdysone‐inducible polypeptides (EIPs) and to commitment to eventual proliferative arrest. Later, the treated cells undergo morphological transformation, cease to proliferate, and develop new enzymatic activities, notably, acetylcholineslerase (AChE) activity. These responses have proven useful as models for studying ecdysone action. Here we report the sensitivity of Kc cells to another important insect developmental regulator—juvenile hormone (JH). We find that JH inhibits some, but not all, aspects of the ecdysone response. When Kc cells are treated with ecdysone in the presence of either natural JHs or synthetic analogues, the morphological and proliferative responses are inhibited and AChE induction is blocked. Most striking is that JHs protect the cells from the rapid proliferative commitment induced by ecdysone alone. The JH effects exhibit reasonable dose‐response curves with half‐maximal responses occurring at very low JH concentrations. Nonetheless, even at high JH concentrations the inhibitory effects are incomplete. It is interesting that EIP induction appears to be refractory to JH. It seems clear that JH is not simply a generalized inhibitor of ecdysone‐induced responses.
Regulatory elements and interactions in the Drosophila 68C glue gene cluster
Authors: Martin Marianne , Mettling Clément , Giangrande Angela , Ruiz Claude , Richards Geoff
Published: May 2005

We reviewed studies on the developmental regulation of the 68C glue gene cluster of Drosophila melanogaster. Extensive transformation analyses of Sgs‐3 have shown that four regions necessary for normal expression can be distinguished. The first ( + 10 to ‐50) contains the transcription start site and TATA motif. This region can be replaced functionally by corresponding sequences from the hsp70 gene, but it is sensitive to point mutations in the TATA sequence. The second region (‐50 to ‐98) contains more than one upstream sequence that, in combination with the other elements, leads to stage and tissue‐specific expression. The third region (centered at ‐600) contains an element that enhances transcript levels some 20‐fold. The final region (between ‐1.65 and ‐2.35 kb) contains elements having modest (twofold to threefold) effects on expression, one of which is contained in the coding sequences of Sgs‐7, a second member of the cluster.
Regulation of larval cuticle protein gene expression in Drosophila melanogaster
Authors: Kimbrell Deborah A. , Tojo Shinichiro J. , Alexander Sherry , Brown Elizabeth E. , Tobin Sara L. , Fristrom James W.
Published: May 2005

Genes that encode 3rd instar larval cuticle proteins (LCP's) of Drosophila melanogaster are located in at least two chromosomal sites. The genes encoding four of the five predominant LCP's are located in a cluster at the chromosomal region 44D. They are organized in pairs that are transcribed divergently, and expressed with different timing during the third larval instar. Towards understanding the basis of gene regulation within the 44D cluster, we have analyzed genetic variants, including the 2‐3 variant, which has an insertion of a copia‐like transposable element, H.M.S. Beagle, within the 44D cluster. The Beagle element appears to inactivate the LCP‐3 gene by inserting into its TATA box, but also may cause the precocious expression of two other LCP genes, LCP‐1 and LCP‐f2, in the cluster. The long terminal repeat (LTR) of the Beagle element apparently contains a sequence, perhaps an enhancer‐like element, which causes altered expression of these genes. We have also investigated the cis‐regulatory elements involved in expression of the LCP‐2 gene in wild‐type larvae. We have identified two upstream regions that may contain separate cisregulatory elements. The region between ‐252 bp and ‐515 bp may be essential for any expression of LCP‐2. Additionally, the region between ‐515 bp and ‐795 bp appears to be required for the normal level of expression of the LCP‐2 gene.