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Biomedical subjects

R H Kessin

Publications and source records attributed to R H Kessin.

At least 19 recordsLinked to original sources

How cellular slime molds evade nematodes.

We have found a predator-prey association between the social amoeba Dictyostelium discoideum and the free soil living nematode Caenorhabditis elegans. C. elegans feeds on the amoebae and multiplies indefinitely when amoebae are the sole food source. In an environment created from soil, D. discoideum grows and develops, but not in the presence of C. elegans. During development, C. elegans feeds on amoebae until they aggregate and synthesize an extracellular matrix called the slime sheath. After the sheath forms, the aggregate and slug are protected. Adult nematodes ingest Dictyostelium spores, which pass through the gut of the worm without loss of structure and remain viable. Nematodes kill the amoebae but disperse the spores. The sheath that is constructed when the social amoebae aggregate and the spore coats of the individual cells may protect against this predator. Individual amoebae may also protect themselves by secreting compounds that repel nematodes.

Animals

The phosphodiesterase secreted by prestalk cells is necessary for Dictyostelium morphogenesis.

Dictyostelium discoideum secretes a cyclic nucleotide phosphodiesterase to control cAMP levels during development. Three promoters control expression of the gene--one during vegetative growth, one during aggregation, and one which constrains phosphodiesterase synthesis to prestalk cells. In this report we show that the expression of phosphodiesterase (PDE) in prestalk cells is necessary for morphogenesis. A gene that codes for a specific glycoprotein inhibitor of the phosphodiesterase (Kd = 0.1 nM) was fused to the prestalk-specific promoter of the PDE gene. Transformants carrying multiple copies of this construct secreted inhibitor in 100-fold excess after the aggregation process had occurred. The first effect seen was an elongated tip, followed by a block in slug formation and an inability to culminate. Stalk and spores cells are produced but morphogenesis is uncoupled from cellular differentiation. Overproduction of inhibitor during earlier stages delayed aggregation, but did not affect fruiting body formation. A phosphodiesterase mutant was transformed with a plasmid that expresses PDE only during aggregation and not in prestalk cells. The defect in aggregation was rescued, but the defect in later development was not. The combined results indicate that PDE expression in prestalk cells is critical to morphogenesis. To ask whether the inhibitor gene under its normal regulation had a role in aggregation or later morphogenesis, it was destroyed by homologous recombination. The loss of the gene did not prevent development under the conditions used.

Amino Acid Sequence

Regulation of Dictyostelium early development genes in signal transduction mutants.

The secreted cyclic nucleotide phosphodiesterase (PDE) and its glycoprotein inhibitor (PDI) are among the first genes expressed when Dictyostelium amoebae begin their development. We used a series of mutants with defects in signal transduction to ask whether cAMP receptors 1 and 3, G alpha2, G beta, adenylyl cyclase (ACA), or the protein kinase A catalytic subunit (PKAcat) are required for the initial appearance or later regulation of the PDE and the PDI transcripts. The PDE gene produces a 1.9-kb transcript during vegetative growth and then a 2.4-kb transcript during the early hours of development. Regulation of the 2.4-kb transcript in CAR1, G alpha2, G beta, and ACA mutants is similar to that of isogenic parental strains, although its level is reduced in strains that carry the CAR1 mutation. CAR1/CAR3 double mutants also produce less PDE transcript, but the PDE gene remains inducible by cAMP. The PKAcat mutant produces the 2.4-kb PDE transcript, but in this mutant the vegetative transcript is retained in development. CAR1 and CAR3 are not required for transcription of the PDI gene, but deleting CAR1 leads to overproduction of the PDI transcript. Induction or repression of the PDI gene does not require G alpha2, G beta, or ACA. PKAcat is required for synthesis of the PDI transcript. The results suggest a two-stage regulation of these early genes through a G alpha2/G beta-independent mechanism and an absolute dependence of PDI on the PKAcat.

Adenylyl Cyclases

Database contamination.

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3',5'-Cyclic-GMP Phosphodiesterases

The role of the cyclic nucleotide phosphodiesterase of Dictyostelium discoideum during growth, aggregation, and morphogenesis: overexpression and localization studies with the separate promoters of the pde.

cAMP acts as a primary signal and is regulated by a secreted cyclic nucleotide phosphodiesterase (PDE) throughout development in Dictyostelium discoideum. Expression of the PDE gene (pde) is controlled by promoters specific to vegetative growth (prV), aggregation (prA), or late development (prL). Promoter-containing regions were individually fused to the pde coding sequence. After transformation multiple copies of each construct led to overexpression of PDE mRNA and enzyme activity with the temporal profile expected of each promoter. Overexpression of PDE from prV and prA altered the timing of aggregation compared to control transformants, but the final morphology was normal. Control transformants showed delayed aggregation compared to nontransformed cells. Cells that overexpressed PDE from prL aggregated like the control transformants, but no fruiting bodies were formed. Individual promoter regions were fused to the beta-galactosidase gene (lacZ). Cells that expressed prA-lacZ were dispersed throughout aggregation fields and mounds. Cells that expressed prL-lacZ were first seen distributed homogeneously throughout tight and tipped mounds. In slugs most of these cells are localized in the anterior region. During culmination, cells that expressed the prL-lacZ construct became incorporated into the stalk and were seen in the upper and lower cups surrounding the spore mass.

2',3'-Cyclic-Nucleotide Phosphodiesterases

Chemotactic sorting to cAMP in the multicellular stages of Dictyostelium development.

Dictyostelium transformants that overproduce the extracellular form of cyclic nucleotide phosphodiesterase and so accumulate a reduced amount of cAMP are blocked in development after aggregation in the form of a tight mound, prior to formation of the apical tip. In such mounds, prespore cell differentiation is repressed, and the apical accumulation of prestalk cells is greatly retarded. When a source of cAMP is placed below the arrested mounds, prestalk cells that would normally migrate in an apical direction instead sort downwards to the substratum. Thus, by acting as the chemoattractant that draws prestalk cells to the apex, cAMP signaling directs the formation of a patterned structure.

Animals

Functional cloning of a Dictyostelium discoideum cDNA encoding GMP synthetase.

A functional cloning procedure has been used to recover a cDNA coding for the GMP synthetase of Dictyostelium discoideum. The enzyme is encoded by a single gene, which is actively transcribed during growth, but not during development. The open reading frame encodes a protein of 718 amino acids with a predicted molecular mass of 79.6 kDa. The Dictyostelium enzyme has extensive homology with the GMP synthetase of Escherichia coli and regional homology to other glutamine amidotransferases.

Amino Acid Sequence

8-Chloroadenosine 3',5'-monophosphate inhibits the growth of Chinese hamster ovary and Molt-4 cells through its adenosine metabolite.

8-Chloroadenosine 3',5'-monophosphate has been reported to inhibit growth of various mammalian cell lines at micromolar concentrations. We have used Chinese hamster ovary cell lines with mutated cyclic AMP-dependent protein kinase or altered cyclic nucleotide metabolism to show that a metabolite, 8-chloroadenosine, is formed in the medium and is the active inhibitor of cell growth in Chinese hamster ovary cells. Adding adenosine deaminase to the Chinese hamster ovary cell growth media removes the inhibition of cell growth attributed to 8-chloroadenosine 3',5'-monophosphate. Adenosine deaminase or dipyridamole also protects Molt-4 lymphoblasts from the growth-inhibitory effects of 8-chloroadenosine 3',5'-monophosphate.

8-Bromo Cyclic Adenosine Monophosphate

Cyclic nucleotide phosphodiesterase of Dictyostelium discoideum and its glycoprotein inhibitor: structure and expression of their genes.

The genes coding for the cyclic nucleotide phosphodiesterase (PD) and the PD inhibitory glycoprotein (PDI) have been cloned and characterized. The PDI gene was isolated as a 1.6 kb genomic fragment, which included the coding sequence containing two small introns and 510 nucleotides of non-translated 5' sequence. From the deduced amino acid sequence we predict a protein with a molecular weight (MW) of 26,000 that, in agreement with previous data, contains 15% cysteine residues. Genomic Southern blot analysis indicates that only one gene encodes the inhibitor. Northern blot analysis shows a single transcript of 0.95 kb. The PDI gene is expressed early in development with little transcript remaining following aggregation. The appearance of PDI mRNA is prevented by the presence of cAMP, but when cAMP is removed the transcript appears within 30 minutes. When cAMP is applied to cells expressing PDI the transcript disappears with a half-life of less than 30 minutes. The PD gene of D. discoideum is transcribed into three mRNAs: a 1.9 kb mRNA specific for growth, a 2.4 kb mRNA specific for aggregation, and a 2.2 kb mRNA specific for late development. The 2.2 kb mRNA is also specific for prestalk cells, and is induced by differentiation-inducing factor. All three mRNAs contain the same coding sequence, and differ only in their 5' non-coding sequences. Each mRNA is transcribed from a different promoter, and by using the chloramphenicol acyltransferase gene as a reporter, we have shown that each promoter displays the same regulation as its cognate mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases

Cyclic AMP responses are suppressed in mammalian cells expressing the yeast low Km cAMP-phosphodiesterase gene.

A genomic DNA fragment from Saccharomyces cerevisiae which contains the SRA5 (=PDE2) gene, coding for a low Km cAMP-phosphodiesterase, was transfected into Chinese hamster ovary cells. Clones carring the cAMP-phosphodiesterase gene were capable of growth in the presence of cholera toxin, which slows the growth of untransfected cells by elevating their cAMP levels. The cholera toxin-resistant transfected cell lines expressed high levels of cAMP-phosphodiesterase mRNA and cAMP-phosphodiesterase activity. Basal intracellular cAMP levels were not significantly affected by the presence of the yeast cAMP-phosphodiesterase gene, but elevation of cAMP levels in response to cholera toxin or prostaglandin E1 was suppressed. Induction of the cAMP-responsive tyrosine aminotransferase promoter by cholera toxin was also blocked in cell lines carrying the yeast cAMP-phosphodiesterase gene. Cholera toxin-resistant transfected cell lines were sensitive to the growth inhibitory effects of N6,02'-dibutyryladenosine 3',5'-monophosphate, which can be used to bypass the effects of the yeast cAMP-phosphodiesterase.

1-Methyl-3-isobutylxanthine

Characterization of the yeast low Km cAMP-phosphodiesterase with cAMP analogues. Applications in mammalian cells that express the yeast PDE2 gene.

The essential interactions between cAMP and the yeast low Km cAMP-phosphodiesterase have been analyzed using cAMP analogues and phosphodiesterase inhibitors. cAMP specificity is conferred by hydrogen bonding at the N-6 and N-7 positions. In contrast to the other yeast phosphodiesterase, (Rp)-adenosine 3',5'-monophosphorothioate is not hydrolyzed. Eleven standard phosphodiesterase inhibitors were not highly effective. In Chinese hamster ovary (CHO) cells that express the yeast cAMP-phosphodiesterase (PDE2) gene, cAMP levels cannot be raised by cholera toxin. cAMP analogues that are efficiently hydrolyzed by the yeast cAMP-phosphodiesterase had no effect on the growth of CHO cells that express the PDE2 gene, even though they block the growth and alter the morphology of control cells. cAMP analogues that are not hydrolyzed by the yeast enzyme inhibited the growth and changed the morphology of both control and PDE2 expressing CHO cells. We have developed a method for creating cell lines in which cAMP levels can be reduced by expression of an exogenous cAMP-phosphodiesterase gene. By employing cAMP analogues that are not hydrolyzed by this phosphodiesterase, the inhibitory effects of the enzyme can be bypassed.

3',5'-Cyclic-AMP Phosphodiesterases

Enzymatic synthesis of the cAMP antagonist (Rp)-adenosine 3',5'-monophosphorothioate on a preparative scale.

(Rp)-Adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) is a highly specific antagonist of the cAMP-dependent protein kinase from eukaryotic cells and is a very poor substrate for phosphodiesterases. It is therefore a useful tool for investigating the role of cAMP as a second messenger in a variety of biological systems. Taking advantage of stereospecific inversion of configuration around the alpha-phosphate during the adenylate cyclase reaction, we have developed a method for the preparative enzymatic synthesis of the Rp diastereomer of adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) from the Sp diastereomer of adenosine 5'-O-(1-thiotriphosphate) ((Sp)-ATP alpha S). The adenylate cyclase from Bordetella pertussis, partially purified by calmodulin affinity chromatography, cyclizes (Sp)-ATP alpha S approximately 40-fold more slowly than ATP, but binds (Sp)-ATP alpha S with about 10-fold higher affinity than ATP. The triethylammonium salt of the reaction product can be purified by elution from a gravity flow reversed-phase C18 column with a linear gradient of increasing concentrations of methanol. Yields of the pure (Rp)-cAMPS product of a synthesis with 2 mg of substrate are about 75%.

Adenylyl Cyclases

The cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum contains three promoters specific for growth, aggregation, and late development.

The cyclic nucleotide phosphodiesterase (phosphodiesterase) plays essential roles throughout the development of Dictyostelium discoideum. It is crucial to cellular aggregation and to postaggregation morphogenesis. The phosphodiesterase gene is transcribed into three mRNAs, containing the same coding sequence connected to different 5' untranslated sequences, that accumulate at different times during the life cycle. A 1.9-kilobase (kb) mRNA is specific for growth, a 2.4-kb mRNA is specific for aggregation, and a 2.2-kb mRNA is specific for late development and is only expressed in prestalk cells. Hybridization of RNA isolated from cells at various stages of development with different upstream regions of the gene indicated separate promoters for each of the three mRNAs. The existence of specific promoters was confirmed by fusing the three putative promoter regions to the chloramphenicol acetyltransferase reporter gene, and the analysis of transformants containing these constructs. The three promoters are scattered within a 4.1-kilobase pair (kbp) region upstream of the initiation codon. The late promoter is proximal to the coding sequence, the growth-specific promoter has an initiation site that is 1.9 kbp upstream of the ATG codon, and the aggregation-specific promoter has an initiation site 3 kbp upstream.

Amino Acid Sequence

Rescue of a Dictyostelium discoideum mutant defective in cyclic nucleotide phosphodiesterase.

One of the developmentally induced gene products that is essential for chemotaxis of Dictyostelium amoebae is a cyclic nucleotide phosphodiesterase. The enzyme can be secreted or exist in a membrane bound form. This enzyme is missing in the mutant HPX235 which, as a consequence, does not aggregate unless exogenous cAMP phosphodiesterase is supplied. We have introduced multiple copies of the cloned phosphodiesterase gene into mutant amoebae and restored aggregation. The formation of anatomically correct fruiting bodies, which does not occur when exogenous enzyme is added, is also restored by transformation with the gene. The construct that we have used gives rise only to secreted phosphodiesterase and therefore the membrane bound form of the enzyme is not absolutely required for normal aggregation and morphogenesis.

3',5'-Cyclic-AMP Phosphodiesterases

The cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum utilizes alternate promoters and splicing for the synthesis of multiple mRNAs.

The cyclic nucleotide phosphodiesterase (phosphodiesterase) gene plays essential roles in the development of Dictyostelium discoideum during cellular aggregation and postaggregation morphogenesis. Genomic clones spanning the gene were isolated and used to determine the sequence and structure of the phosphodiesterase gene. We found an unusually complex organization for a gene of D. discoideum. Two transcripts of 2.4 and 1.9 kilobases (kb) were synthesized from start sites separated by 1.1 kb. A developmentally regulated promoter was utilized for the 2.4-kb mRNA, and a constitutive promoter regulated synthesis of the 1.9-kb transcript. The gene was found to be divided into four exons that are alternately spliced to give rise to the two mRNAs. The precursor of the 2.4-kb mRNA contained a 2.3-kb intron, whereas the precursor of the constitutive transcript was synthesized with a 1.7-kb intron. The two transcripts contained identical protein-coding regions and 400-nucleotide 3' untranslated sequences. The 2.4-kb developmentally regulated mRNA was distinguished by a long 5' untranslated leader of 666 nucleotides. The complex structure of the gene may allow multiple levels of control of the expression of the phosphodiesterase during development.

Base Sequence

The cyclic nucleotide phosphodiesterase of Dictyostelium discoideum: the structure of the gene and its regulation and role in development.

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 single-phosphodiesterase 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 pdsA gene and four fgd 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.

3',5'-Cyclic-AMP Phosphodiesterases