A genetic interaction between a ubiquitin-like protein and ubiquitin-mediated proteolysis in Dictyostelium discoideum(1).

A ubiquitination factor, NosA, is essential for cellular differentiation in Dictyostelium discoideum. In the absence of nosA, development is blocked, resulting in a developmental arrest at the tight-aggregate stage, when cells differentiate into two precursor cell types, prespore and prestalk cells. Development is restored when a second gene, encoding the ubiquitin-like protein SonA, is inactivated in nosA-mutant cells. SonA has homology over its entire length to Dsk2 from Saccharomyces cerevisiae, a ubiquitin-like protein that is involved in the assembly of the spindle pole body. Dsk2 and SonA are both stable proteins that do not seem to be subjected to degradation via the ubiquitin pathway. SonA does not become ubiquitinated and the intracellular levels of SonA are not affected by the absence of NosA. The high degree of suppression suggests that SonA rescues most or all of the defects caused by the absence of nosA. We propose that NosA and SonA act in concert to control the activity of a developmental regulator that must be deactivated for cells to cross a developmental boundary.

Dictyostelium amoebae lacking an F-box protein form spores rather than stalk in chimeras with wild type.

Using a selection for Dictyostelium mutants that preferentially form spores, we have recovered a mutant called CheaterA. In chimeras with isogenic wild-type cells, the CheaterA mutant preferentially forms viable spores rather than inviable stalk cells. The mutant causes wild-type cells that have begun to express spore-specific genes to accumulate in the prestalk compartment of the developing organism. In the wild-type cells, the chtA transcript is absent in growing cells and appears early in development. No transcript was detected in the mutant by Northern blot. The chtA gene codes for a protein with an F-box and WD40 domains. This class of protein usually forms part of an Skp1, cullin, F-box (SCF) complex that targets specific protein substrates for ubiquitination and degradation.

The characterization of two Dictyostelium discoideum genes encoding ribosomal proteins with sequence similarity to rat L27a and L37a.

Two Dictyostelium discoideum ribosomal protein genes, denoted DdL27a and DdL37a, were isolated and sequenced. The DdL27a gene contained an open reading frame of 148 amino acids coding for a putative 16,407 Da protein, which was similar to rat L27a (82.6% similarity) and to ribosomal proteins from other species. The gene contained a 311-bp intron downstream from the ATG initiation codon with an A+T content of 75%. The DdL37a gene encoded a 9,999 Da protein consisting of 91 amino acids, which had high sequence similarity to rat, human, and chicken ribosomal protein L37a, and was interrupted by two introns of 254 bp and 75 bp in length. The DdL37a protein contained a typical zinc finger motif (Cys-X2-Cys-X14-Cys-X2-Cys), which may be involved in the interaction of proteins with nucleic acids. Genomic DNA blot analysis indicated that the DdL27a and DdL37a genes are present in single copies in the Dictyostelium haploid genome. The DdL27a and DdL37a mRNA were expressed maximally in growing amoebae, and their levels decreased during multicellular development, coordinately with the observed decrease in ribosome accumulation during later development.

Characterization of the Dictyostelium discoideum cellulose-binding protein CelB and regulation of gene expression.

Similar to other stages of Dictyostelium development, spore germination is a particularly suitable model for studying regulation of gene expression. The transition from spore to amoeba is accompanied by developmentally regulated changes in both protein and mRNA synthesis. A number of spore germination-specific cDNAs have been isolated previously. Among these are two members of the 270 gene family, a group of four genes defined by the presence of a common tetrapeptide repeat of Thr-Glu-Thr-Pro. celA (formerly called 270-6) and celB (formerly 270-11) are expressed solely and coordinately during spore germination, the levels of the respective mRNAs being low in dormant spores, rising during germination to a maximum level at about 2 h, and then rapidly declining as amoebae are released from spores. The mRNAs are not found in growing cells or during multicellular development. The rapidity with which these transcripts accumulate and then disappear during germination implies that the respective products may be important for the process. We reported previously that the CelA protein is a cellulase (endo-1, 4-beta-glucanase (EC 3.2.1.4)). In the present investigation, properties of the CelB protein, a glycosylated protein of 532 amino acids, 36% of which are serine or threonine, were examined, and the upstream sequences involved in the developmental regulation of the expression of the gene have been determined. The CelB protein does not demonstrate cellulase activity, but it has a cellulose-binding domain. Its role, if any, in degradation of the cellulose-containing spore wall is unknown. To identify cis-acting elements in the celB promoter, unidirectional 5' deletions of the celB upstream noncoding region were constructed and used to transform amoebae. Analysis of promoter activity during different stages of development shows that a short, very A/T-rich sequence of approximately 81 base pairs is sufficient for spore-specific celB transcription. Contained in this sequence is the Myb oncogene protein binding site, TAACTG, which was shown previously to be a negative regulator of celA transcription. Dictyostelium and mouse Myb proteins bind to this region of the promoter, suggesting that Myb might regulate celB gene expression negatively as it does in celA.

Isolation of a Dictyostelium discoideum 14-3-3 homologue.

A 1.0 kb cDNA clone (Dd14-3-3) encoding a 14-3-3 homologue was isolated from a Dictyostelium discoideum cDNA library. The putative Dd14-3-3 protein has highest sequence identity to a barley 14-3-3 isoform (74%). Southern blot analysis suggests that only one 14-3-3 gene is present in the Dictyostelium genome. Highest Dd14-3-3 expression is observed in vegetatively growing cells, and expression decreases during multicellular development. In contrast, Dd14-3-3 protein levels detected immunochemically remained constant during Dictyostelium development. Expression of the Dd14-3-3 cDNA in Saccharomyces cerevisiae complemented the lethal disruption of the two yeast genes encoding 14-3-3 proteins (BMH1 and BMH2). This shows that Dd14-3-3 can fulfil the same function(s) as the yeast 14-3-3 proteins.

AT-rich upstream sequence elements regulate spore germination-specific expression of the Dictyostelium discoideum celA gene.

Two members of a family of spore germination-specific cDNAs, celA and celB, are expressed coordinately, exclusively during spore germination. In the present study the regulatory sequence elements responsible for celA germination-specific expression have been identified. The very AT-rich 81 bp sequence between -664 and -584 upstream of the translation initiation site was required for proper temporal transcription of the celA gene. This sequence is comprised of two cis elements, each of which was active by itself in allowing celA expression. Electrophoretic mobility shift assays showed that a factor(s) in an extract prepared from germinating spores bound to the celA regulatory region. One of the three complexes formed was specific for the germinating spore extract. The results are consistent with the notion that the factor(s) that binds to this regulatory region is involved in expression of celA.

Cloning and functional expression of a Dictyostelium discoideum protein tyrosine phosphatase.

Using polymerase chain reaction methods, we cloned a 1.7-kilobase cDNA, denoted DdPTPa, that has high homology with other known eukaryotic protein tyrosine phosphatases. DdPTPa possess a 241-amino acid protein tyrosine phosphatase domain located in the C terminus, which exhibits a 39-43% amino acid sequence identity with published protein tyrosine phosphatases. Absence of a characteristic signal sequence and transmembrane domain suggests that DdPTPa is a nonreceptor type cytoplasmic protein tyrosine phosphatase. Southern blot analysis of genomic DNA indicates the presence of a multigene protein tyrosine phosphatase family in Dictyostelium. Northern blot analysis reveals four species of mRNA that hybridize to the DdPTPa probe, at least three of which are developmentally regulated. The entire coding sequence of DdPTPa was subcloned into the pET15-b vector and expressed in Escherichia coli. Affinity-purified DdPTPa protein efficiently dephosphorylates both p-nitrophenyl phosphate and tyrosine-phosphorylated reduced, carboxyamidomethylated, and maleylated lysozyme. A Dictyostelium transformant overexpressing DdPTPa does not develop normally. The overexpresser fails to aggregate, in contrast to the control transformant containing vector alone, and after 24 h gives rise to only a few abnormal slugs and small fruiting bodies.

The highly divergent alpha- and beta-tubulins from Dictyostelium discoideum are encoded by single genes.

As a step in the characterization of the microtubule system of Dictyostelium discoideum, we have isolated and sequenced full-length cDNA clones that encode the Dictyostelium alpha- and beta-tubulins, as well as the Dictyostelium alpha-tubulin gene. Southern blot analysis suggests that Dictyostelium is unusual in that its genome contains single alpha- and beta-tubulin genes, rather than the multi-gene family common in most eukaryotic organisms. The complete alpha-tubulin cDNA contains 1558 nucleotides, with an open reading frame, that encode a protein of 457 amino acids. The complete beta-tubulin cDNA contains 1572 nucleotides and encodes a protein of 456 amino acids. Analysis of the deduced protein sequences indicates that while there is a significant degree of sequence similarity between the Dictyostelium tubulins and other known tubulins, the Dictyostelium alpha-tubulin displays the greatest sequence divergence yet described. Single alpha- and beta-tubulin transcripts are detected by northern blot analysis during all stages of Dictyostelium development. The highest levels of message accumulate late in germinating spores and vegetative amoebae. Despite changes in alpha- and beta-tubulin mRNA levels, protein levels remain constant throughout development. We have expressed the carboxy-terminal two-thirds of the alpha- and beta-tubulins as trpE fusions in Escherichia coli and used this protein to produce polyclonal antisera specific for the Dictyostelium alpha- and beta-tubulins. These antisera recognize one alpha- and two beta-tubulin spots on western blots of 2-D gels and, by indirect immunofluorescence, both recognize the interphase and mitotic microtubule arrays in vegetative amoebae.

Molecular cloning and developmental regulation of Dictyostelium discoideum homologues of the human and yeast HIV1 Tat-binding protein.

TBP1 and 7, two human cDNA clones, are members of a large, highly conserved family that encode proteins that bind to type 1 human immunodeficiency virus Tat protein, and may be transcription factors. Two Dictyostelium discoideum homologues, denoted DdTBP2 and 10, were isolated and characterized, and these have striking sequence similarity with both the human and yeast counterparts of the family. RNA blot analysis indicates that both DdTBP2 and 10 are developmentally regulated. Transcripts representing these cDNAs are highest in vegetatively growing cells, and their levels fall steadily throughout multicellular development and are not found in dormant or germinating spores. If indeed these proteins in Dictyostelium are also transcription factors, then this finding might indicate that only certain developmentally regulated genes require them.

The Dictyostelium discoideum spore germination-specific cellulase is organized into functional domains.

During Dictyostelium discoideum spore germination, degradation of the cellulose-containing spore wall is required to allow the amoeba to emerge. The CelA gene, which is transcribed and expressed exclusively during spore germination, codes for a 705-amino-acid protein that has cellulase activity [endo-(1,4)-beta-D-glucanase]. Amoebae transformed by a vector containing the CelA coding sequence or portions of it transcribed from a heterologous promoter expressed and secreted full-length or suitably truncated proteins during vegetative growth when, under normal conditions, these proteins are not made. The gene constructs divided the CelA protein into three domains: a 461-amino-acid N-terminal region that has significant similarity to those of other cellulases and that has been shown to be the catalytic domain; a contiguous 91-residue repeat containing the motif threonine-glutamic acid-threonine-proline, which is glycosylated; and, joined to the repeat, a C-terminal 153-amino-acid sequence that most probably defines a cellulose-binding domain.

Biological and molecular correlates between induced dedifferentiation and spore germination in Dictyostelium.

When developing cultures of Dictyostelium discoideum are disaggregated at any time prior to cell wall formation and challenged to reinitiate development, amoebae will progress through the original sequence of morphogenetic stages, but the second time through they will do so in roughly one-tenth the original time, a process known as 'rapid recapitulation'. However, if disaggregated cells are suspended in nutrient medium, they enter a program of dedifferentiation during which they lose the capacity to rapidly recapitulate after an 80 minute lag period in a process known as 'erasure'. Here we show that cells that have completed the morphogenetic program and emerge from spore coats in the process of germination have also erased. In addition, the germination-specific 270 gene family is expressed during induced dedifferentiation in a unique fashion, and a germination-defective mutant exhibits a dramatic delay in erasure without concomitant defects in the program of gene regulation accompanying induced dedifferentiation. These results suggest for the first time that induced dedifferentiation and spore germination share some common processes in converting cells from a developmental to vegetative state.

A Dictyostelium discoideum cellulase is a member of a spore germination-specific gene family.

A member of the 270 spore germination-specific gene family in Dictyostelium discoideum is shown to encode a cellulase (endo-(1,4)-beta-D-glucanase, EC 3.2.1.4) activity. The 270-6 deduced protein shows 38% identity and 58% similarity to an avocado (Persea americana) cellulase. During spore germination in Dictyostelium extracellular cellulase activity starts to accumulate coincident with the appearance of the 270-6 gene transcript. Amoebae transformed by a vector containing the 270-6 mRNA sequence express an extracellular cellulase during vegetative growth when there would otherwise be no cellulase activity. In addition, the expression of a truncated 270-6 polypeptide lacking the 270 gene family-defining tetrapeptide repeat and the C-terminal region, in suitably transformed amoebae, also produces an extracellular cellulase activity. Several differently sized cellulase activities are shown to accumulate during spore germination, and it is possible that the 270 gene family represents a coordinately expressed family of cellulases.

Dictyostelium discoideum gene family contains a long internal amino acid repeat.

Two different cDNA clones denoted pTO270-6 and pTO270-11 represent two mRNAs that are developmentally regulated during spore germination in Dictyostelium discoideum. The respective mRNAs are found only during early germination and are not present in other stages of growth or multicellular development. Four different genomic clones that hybridize to sequences that are common to both of the 270 cDNA clones were isolated from Dictyostelium libraries and sequenced. Two are the genes for the two cDNAs, and the other two represent genes that do not seem to be transcribed. All four genomic sequences possess a very unusual internal feature in the deduced protein sequences composed of a monotonous repeat of the tetrapeptide threonine-glutamic acid-threonine-proline. The other portions of the proteins have no homology among themselves. The deduced protein corresponding to the 270-6 gene is very similar to avocado (Persea americana) cellulase. Since cellulose in the spore wall has to be digested during spore germination this suggests that this protein may function as an endo-(1,4)-beta-D-glucanase during germination.

A shared internal threonine-glutamic acid-threonine-proline repeat defines a family of Dictyostelium discoideum spore germination specific proteins.

A cDNA denoted pRK270 hybridizes to two mRNA species in RNA blots. The mRNAs specific to this clone are not expressed during vegetative growth and multicellular development. They are, however, found predominantly during early stages of spore germination, suggesting that their synthesis is rapidly and coordinately turned on during germination. Two different cDNAs named 270-6 and 270-11 were isolated, representing the two mRNAs. DNA blot analysis shows that 270 is a multigene family. Four genes were isolated from Dictyostelium genomic libraries and sequenced. The putative proteins coded for by these genes are about 51,000, 55,000, 76,000, and 100,000 Da. Two of the genes are expressed during spore germination while transcripts for the other two are not present during spore germination, vegetative growth, or the stages of multicellular development studied. The cDNAs and genes code for deduced proteins that possess a very unusual internal amino acid repeat comprised of the tetrapeptide threonine-glutamic acid-threonine-proline. The other portions of the proteins have no homology among themselves. The 270-6 protein shows excellent identity with avocado (Persea americana) cellulase, indicating that it may function as an endo-(1,4)-beta-D-glucanase.

Nucleotide sequences of Dictyostelium discoideum developmentally regulated cDNAs rich in (AAC) imply proteins that contain clusters of asparagine, glutamine, or threonine.

A Dictyostelium discoideum repetitive element composed of long repeats of the codon (AAC) is found in developmentally regulated transcripts. The concentration of (AAC) sequences is low in mRNA from dormant spores and growing cells and increases markedly during spore germination and multicellular development. The sequence hybridizes to many different sized Dictyostelium DNA restriction fragments indicating that it is scattered throughout the genome. Four cDNA clones isolated contain (AAC) sequences in the deduced coding region. Interestingly, the (AAC)-rich sequences are present in all three reading frames in the deduced proteins, i.e., AAC (asparagine), ACA (threonine) and CAA (glutamine). Three of the clones contain only one of these in-frame so that the individual proteins carry either asparagine, threonine, or glutamine clusters, not mixtures. However, one clone is both glutamine- and asparagine-rich. The (AAC) portion of the transcripts are reiterated 300 times in the haploid genome while the other portions of the cDNAs represent single copy genes, whose sequences show no similarity other than the (AAC) repeats. The repeated sequence is similar to the opa or M sequence found in Drosophila melanogaster notch and homeo box genes and in fly developmentally regulated transcripts. The transcripts are present on polysomes suggesting that they are translated. Although the function of these repeats is unknown, long amino acid repeats are a characteristic feature of extracellular proteins of lower eukaryotes.

Molecular organization of developmentally regulated Dictyostelium discoideum ubiquitin cDNAs.

Dictyostelium discoideum ubiquitin mRNAs are regulated in a complex fashion during spore germination and multicellular development. Species of mRNA of 1900, 1400, 1100, 840, 580, and 500 nucleotides (nt) are found which are expressed differentially during different stages of development. DNA blot analysis indicates that ubiquitin genes constitute a multigene family of at least six genes. cDNAs representing all the ubiquitin mRNA transcripts were isolated and sequenced. The Dictyostelium mRNAs are organized as tandem repeats of the 76 amino acid ubiquitin unit (228 nt). We isolated one cDNA containing seven of these tandem repeats, and two different five- and three-repeat cDNAs. In addition, 2 cDNAs containing a single ubiquitin repeat fused at its 3' end to an unrelated 52 and 78 amino acid extension were identified. There is a remarkable similarity in the sequences of the non-ubiquitin extensions among yeast and mammalian counterparts. The extensions are very basic, containing approximately 30% lysine/arginine. Another common feature of these proteins is the presence of a common structural motif containing cysteine residues at conserved positions, suggesting a metal binding domain that matches a consensus sequence of Xenopus transcription factor TFIIIA and other nucleic acid binding proteins. The characterization of ubiquitin cDNAs and genomic sequences in D. discoideum now makes the understanding of its developmental regulation feasible.

Organization of a gene family developmentally regulated during Dictyostelium discoideum spore germination.

mRNA specific to cDNA clone pLK109 is present in Dictyostelium discoideum spores, increases about two- to threefold at 0.5 to 1 h during spore germination, and then rapidly decreases. The mRNA is not detectable in vegetative cells or in early multicellular development on filters, but is present late during development, approximately at the time of sporulation. 109 mRNA in spores is 700 nucleotides in length but this is processed during germination by shortening of the poly(A) tail to about 600 nucleotides at 1 to 1.5 hours. pLK109 is a member of a multigene family containing three separate genes, and we have isolated and sequenced all of them. All three sequences code for deduced proteins of 127 amino acid residues, with only a few amino acid differences among them. Gene 1 represents the "transcribed" gene, since all 33 cDNAs we isolated are identical with the cDNA pLK109 and the coding region of this gene. Other open reading frames are in close proximity to each of the 109 sequences. About 200 base-pairs 3' to the gene 1 109 sequence is an open reading frame in the opposite orientation. Gene 2 fragment contains a sequence that codes for a protein similar to trypanosome alpha-tubulin 728 base-pairs 5' to the 109 sequence. Gene 3 fragment possesses two additional putative coding regions, one 5' and another 3' to the 109 gene. There is a remarkable similarity between the 5' upstream regions of all three genes. Each possesses a normal Dictyostelium TATA box and the usual T stretch. In addition, there are many other portions of about 400 to 500 base-pairs of the 5' regions that are either identical for long stretches or very similar.

Characterization of genes that are developmentally regulated during Dictyostelium discoideum spore germination.

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.

Structure of two developmentally regulated Dictyostelium discoideum ubiquitin genes.

A previously isolated cDNA clone, pLK229, that is specific for mRNA developmentally expressed during Dictyostelium discoideum spore germination and multicellular development, was used to screen two genomic libraries. Two genomic sequences homologous to pLK229 were isolated and sequenced. Genomic clone p229 is identical to the cDNA clone pLK229 and codes for a polypeptide of 381 amino acids. This polypeptide is composed of five tandem repeats of the same 76-amino-acid sequence. Clone lambda 229 codes for a protein of 229 amino acids, containing three tandem repeats of the identical 76-amino-acid sequence. A computer search for homology to known proteins revealed that the 76-amino-acid repeat was identical to human and bovine ubiquitin except for two amino acid differences.

Role of protein synthesis in decay and accumulation of mRNA during spore germination in the cellular slime mold Dictyostelium discoideum.

Spore germination in Dictyostelium discoideum is a particularly suitable model for studying the regulation of gene expression, since developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. The previous isolation of three cDNA clones specific for mRNA developmentally regulated during spore germination allowed for the quantitation of the specific mRNAs during this process. The three mRNAs specific to clones pLK109, pLK229, and pRK270 have half-lives much shorter (minutes) than those of constitutive mRNAs (hours). Using spore germination as a model, we studied the roles of ribosome-mRNA interactions and protein synthesis in mRNA degradation by using antibiotics that inhibit specific reactions in protein biosynthesis. Cycloheximide inhibits the elongation step of protein synthesis. Polysomes accumulate in inhibited cells because ribosomes do not terminate normally and new ribosomes enter the polysome, eventually saturating the mRNA. Pactamycin inhibits initiation, and consequently polysomes break down in the presence of this drug. Under this condition, the mRNA is essentially free of ribosomes. pLK109, pLK229, and pRK270 mRNAs were stabilized in the presence of cycloheximide, but pactamycin had no effect on their normal decay. Since it seems likely that stability of mRNA reflects the availability of sites for inactivation by nucleases, it follows that in the presence of cycloheximide, these sites are protected, presumably by occupancy by ribosomes. No ribosomes are bound to mRNA in the presence of pactamycin, and therefore mRNA degrades at about the normal rate. The data further indicate that a labile protein is probably not involved in mRNA decay or stabilization, since protein synthesis is inhibited equally by both antibiotics. We conclude that it may be important to use more than one type of protein synthesis inhibitor to evaluate whether protein synthesis is required for mRNA decay. The effect of protein synthesis inhibition on mRNA synthesis and accumulation was also studied. mRNA synthesis continues in the presence of inhibitors, albeit at a diminished rate relative to that of the uninhibited control.