Multiple cellular processes affected by the absence of the Rpb4 subunit of RNA polymerase II contribute to the deficiency in the stress response of the yeast rpb4(delta) mutant.

We previously described the isolation of yeast mutants (sex mutants) that secrete reduced amounts of mature alpha-factor when it is synthesized as part of a fusion with prosomatostatin. In the present study we show that the sex3-1 mutant displays pleiotropic phenotypes. These include an abnormal morphology, an osmoremediable caffeine sensitivity, reduced secretion of mature alpha-factor, a weakened cell wall and a marked deficiency in halotolerance. Cloning of the SEX3 gene revealed that it is identical to the RPB4 gene. This gene encodes the fourth largest subunit of yeast RNA polymerase II, which has been postulated to play a major role in the response to stress. We show that transcriptional activation in response to either a cell wall stress or to growth in the presence of elevated salt concentrations is minimally affected by the loss of RPB4 function. However, whereas the levels of several mRNAs are similarly reduced (by about 30%) in rpb4 mutants grown in rich medium at moderate temperature, some transcripts, in particular ZDS1, are more abundant. An increase dosage of ZDS1, or of genes involved in cell wall assembly and in secretion (RHO1 and SR077, respectively), partially suppresses the sensitivity of rpb4delta cells to high temperature, heat shock and stationary phase. Collectively, our results indicate that the loss of Rpb4p perturbs several cellular functions that contribute to the inappropriate stress response of rpb4delta yeast. We therefore conclude that this RNA poiymerase II subunit is not specifically involved in the stress response.

Suppression of the profilin-deficient phenotype by the RHO2 signaling pathway in Saccharomyces cerevisiae.

Profilin plays an important role in actin organization in all eukaryotic cells through mechanisms that are still poorly understood. We had previously shown that Mid2p, a transmembrane protein and a potential cell wall sensor, is an effective multicopy suppressor of the profilin-deficient phenotype in Saccharomyces cerevisiae. To better understand the role of Mid2p in the organization of the actin cytoskeleton, we isolated five additional multicopy suppressors of pfy1Delta cells that are Rom1p, Rom2p, Rho2p, Smy1p, and the previously uncharacterized protein Syp1p. The problems of caffeine and NaCl sensitivity, growth defects at 30 degrees and 37 degrees, the accumulation of intracellular vesicular structures, and a random budding pattern in pfy1Delta cells are corrected by all the suppressors tested. This is accompanied by a partial repolarization of the cortical actin patches without the formation of visible actin cables. The overexpression of Mid2p, Rom2p, and Syp1p, but not the overexpression of Rho2p and Smy1p, results in an abnormally thick cell wall in wild-type and pfy1Delta cells. Since none of the suppressors, except Rho2p, can correct the phenotype of the pfy1-111/rho2Delta strain, we propose a model in which the suppressors act through the Rho2p signaling pathway to repolarize cortical actin patches.

The one-kilobase DNA fragment upstream of the ardC actin gene of Physarum polycephalum is both a replicator and a promoter.

The 1-kb DNA fragment upstream of the ardC actin gene of Physarum polycephalum promotes the transcription of a reporter gene either in a transient-plasmid assay or as an integrated copy in an ectopic position, defining this region as the transcriptional promoter of the ardC gene (PardC). Since we mapped an origin of replication activated at the onset of S phase within this same fragment, we examined the pattern of replication of a cassette containing the PardC promoter and the hygromycin phosphotransferase gene, hph, integrated into two different chromosomal sites. In both cases, we show by two-dimensional agarose gel electrophoresis that an efficient, early activated origin coincides with the ectopic PardC fragment. One of the integration sites was a normally late-replicating region. The presence of the ectopic origin converted this late-replicating domain into an early-replicating domain in which replication forks propagate with kinetics indistinguishable from those of the native PardC replicon. This is the first demonstration that initiation sites for DNA replication in Physarum correspond to cis-acting replicator sequences. This work also confirms the close proximity of a replication origin and a promoter, with both functions being located within the 1-kb proximal region of the ardC actin gene. A more precise location of the replication origin with respect to the transcriptional promoter must await the development of a functional autonomously replicating sequence assay in Physarum.

Overexpression of MID2 suppresses the profilin-deficient phenotype of yeast cells.

Profilin-deficient Saccharomyces cerevisiae cells show abnormal growth, actin localization, chitin deposition, bud formation and cytokinesis. Previous studies have also revealed a synthetic lethality between pfy1 and late secretory mutants, suggesting a role for profilin in intracellular transport. In this work, we document further the secretion defect associated with the pfy1delta mutant. Electron microscopic observations reveal an accumulation of glycoproteins in the bud and in the mother cell. The MATa, pfy1delta cells mate as well as wild-type cells, while the mating efficiency of MAT alpha, pfy1delta cells is reduced. Pulse-chase experiments demonstrate an accumulation of the 19 kDa alpha-factor precursor and delayed secretion of the mature alpha-factor. The TGN protein Kex2p is the principal enzyme responsible for the endoproteolytic cleavage of the alpha-factor precursor. An immunofluorescence detection of Kex2p shows an altered localization in pfy1delta cells. Instead of a discrete punctate distribution, the enzyme is dispersed throughout the cytoplasm. A high-copy-number plasmid containing MID2, which encodes a potential transmembrane protein involved in cell cycle control, suppresses the abnormal growth, actin distribution, alpha-factor maturation and the accumulation of intracellular membranous structures in pfy1delta cells.

Mapping of a replication origin within the promoter region of two unlinked, abundantly transcribed actin genes of Physarum polycephalum.

We analyzed the replication of two unlinked actin genes, ardB and ardC , which are abundantly transcribed in the naturally synchronous plasmodium of the slime mold Physarum polycephalum. Detection and size measurements of single-stranded nascent replication intermediates (RIs) demonstrate that these two genes are concomitantly replicated at the onset of the 3-h S phase and tightly linked to replication origins. Appearance of RIs on neutral-neutral two-dimensional gels at specific time points in early S phase and analysis of their structure confirmed these results and further established that, in both cases, an efficient, site-specific, bidirectional origin of replication is localized within the promoter region of the gene. We also determined similar elongation rates for the divergent replication forks of the ardC gene replicon. Finally, taking advantage of a restriction fragment length polymorphism, we studied allelic replicons and demonstrate similar localizations and a simultaneous firing of allelic replication origins. Computer search revealed a low level of homology between the promoters of ardB and ardC and, most notably, the absence of DNA sequences similar to the yeast autonomously replicating sequence consensus sequence in these Physarum origin regions. Our results with the ardB and ardC actin genes support the model of early replicating origins located within the promoter regions of abundantly transcribed genes in P. polycephalum.

The two alleles of the hapP gene in Physarum polycephalum code for different proteins.

Many mRNAs show cell-type specific expression in the acellular slime mold Physarum polycephalum. The most abundant plasmodial-specific mRNA (hapP) encodes a small hydrophobic protein of 187 amino acids that contains a potential signal peptide. Southern hybridizations using the hapP cDNA showed that the hapP gene is a single copy gene with two alleles, hapP1 and hapP2. The alleles have restriction enzyme polymorphisms. The nucleotide sequence of the coding region of the hapP1 allele was obtained from a genomic clone, and the nucleotide sequence of the hapP2 allele was obtained from a cDNA clone. The hapP1 and hapP2 alleles code for proteins that are 9.6% different in amino acid sequence. All differences are found in the central region of the protein. The nucleotide sequences of the first and last exons, which contain coding and non-coding regions, are identical. PCR amplification of cDNAs (RT-PCR) showed that both alleles are expressed in the same cell.

Homologous gene replacement in Physarum.

The protist Physarum polycephalum is useful for analysis of several aspects of cellular and developmental biology. To expand the opportunities for experimental analysis of this organism, we have developed a method for gene replacement. We transformed Physarum amoebae with plasmid DNA carrying a mutant allele, ardD delta 1, of the ardD actin gene; ardD delta 1 mutates the critical carboxy-terminal region of the gene product. Because ardD is not expressed in the amoeba, replacement of ardD+ with ardD delta 1 should not be lethal for this cell type. Transformants were obtained only when linear plasmid DNA was used. Most transformants carried one copy of ardD delta 1 in addition to ardD+, but in two (5%), ardD+ was replaced by a single copy of ardD delta 1. This is the first example of homologous gene replacement in Physarum. ardD delta 1 was stably maintained in the genome through growth, development and meiosis. We found no effect of ardD delta 1 on viability, growth, or development of any of the various cell types of Physarum. Thus, the carboxy-terminal region of the ardD product appears not to perform a unique essential role in growth or development. Nevertheless, this method for homologous gene replacement can be applied to analyze the function of any cloned gene.

A proline-rich protein, verprolin, involved in cytoskeletal organization and cellular growth in the yeast Saccharomyces cerevisiae.

A gene (VRP1) encoding a novel proline-rich protein (verprolin) has been isolated from the yeast Saccharomyces cerevisiae as a result of its hybridization to a chick vinculin cDNA probe. The deduced protein sequence contains 24% proline residues present as proline-rich motifs throughout the verprolin sequence. Several of these motifs resemble recently identified sequences shown to bind Src homology 3 (SH3) domains in vitro. Replacement of the wild-type VRP1 allele with a mutant allele results in strains that grow slower than wild-type strains and are temperature sensitive. The vrp1 mutants are impaired in both cell shape and size and display aberrant chitin and actin localization. We propose that verporlin is involved in the maintenance of the yeast actin cytoskeleton, through interactions with other proteins, possibly containing SH3 domains.

Stable, selectable, integrative DNA transformation in Physarum.

The Physarum polycephalum actin promoter, PardC, can drive transient expression of heterologous genes in Physarum amoebae. The hph gene, encoding hygromycin (Hy) phosphotransferase, can confer resistance to Hy on a broad spectrum of organisms. When PardC is translationally fused to hph and transformed into yeasts on high-copy-number vectors, the yeasts become Hy resistant (HyR), showing that PardC-hph is a functional, selectable genetic element. To establish a stable transformation system for Physarum, we electroporated plasmids bearing PardC-hph into Physarum amoebae and then selected for HyR transformants. We show that HyR amoebae arise upon the stable integration of PardC-hph into the nuclear genome in single copy. These results establish a transformation system that can be used to add plasmid-borne genetic information to Physarum.

Two developmentally regulated mRNAs encoding actin-binding proteins in Physarum polycephalum.

A cDNA library from amoebae of Physarum polycephalum was screened by differential hybridization. Two clones contained inserts for mRNAs present in amoebae and absent in plasmodia. The LAV3-4 cDNA encodes a 402 aa protein (ABP-46) that shows sequence similarity to the actin binding site in the N-terminal region of the alpha-actinin family. The LAV3-5 cDNA is 76% identical to the Dictyostelium actin bundling protein, which cross-links and stabilizes actin filaments in amoebal filopodia.

Structure and identity of a late-replicating and transcriptionally active gene.

Eukaryotic genes are usually replicated early during S-phase in the cell lineages in which they are expressed. Using partially characterized cDNA probes, we recently established two exceptions to this rule in the slime mold Physarum polycephalum. In this paper, we analyzed the structure and the identity of one of these two genes. By genomic cloning and Southern analysis we demonstrate that it is a single-copy gene and decipher the structure of the two alleles by taking advantage of a restriction fragment length polymorphism. By cDNA cloning and sequencing, we deduced the amino acid coding capacity of the mRNA. Finally, we confirmed the late replication of this abundantly expressed gene by "gene dosage" analysis, an experiment that did not require any drug treatment of the cell. Our results provide for the characterization and the structure of the first developmentally regulated gene known to be replicated late in S-phase and abundantly expressed within a eukaryotic cell.

Transient expression in Physarum of a chloramphenicol acetyltransferase gene under the control of actin gene promoters.

We cloned and sequenced two actin promoters from Physarum, and constructed plasmids carrying these promoters upstream of a bacterial chloramphenicol acetyltransferase (cat) gene. We then tested the plasmids for their ability to express cat in Physarum amoebae. We present reliable methods for introducing plasmid DNA into Physarum amoebae by electroporation, and show that expression of the cat gene in amoebae occurs in the presence, but not the absence, of one or the other Physarum actin promoter.

An unusual actin-encoding gene in Physarum polycephalum.

Actin is one of the most conserved proteins in eukaryotic organisms. In the present work, we cloned and determined the nucleotide sequence of an unusual actin-encoding gene, ardD, from the slime mold, Physarum polycephalum. The ardD gene encodes an ArdD protein containing 367 amino acids (aa) instead of the 375-376 aa found in a typical actin. The nine missing aa are accounted for by deletions of three aa in the first exon, five in the fifth exon and one in the sixth exon. These deletions in the coding sequence were observed in a polymerase chain reaction (PCR)-generated cDNA fragment, which excludes the possibility of a cloning artifact. In addition, ArdD contains numerous aa substitutions distributed throughout the protein. The ArdD aa sequence was compared with published actin sequences. The most identity is seen with the P. polycephalum ArdA, ArdB and ArdC (84%) and Acanthamoeba (82%) actins, while the least identity is found with Tetrahymena actin (67%). The expression of the ardD gene is developmentally regulated. The highest levels of ardD mRNA were found in spherules, less was seen in plasmodia and no detectable transcripts were observed in amoebae. The PCR amplification of an ardD cDNA from spherules confirmed the presence of mRNA in this developmental stage. The aa deletions and substitutions in the predicted ArdD aa sequence make it one of the most distinctive actins known.

Fission yeast promoter-probe vectors based on hygromycin resistance.

We have constructed fission yeast vectors that carry either complete or 5'-truncated alleles of the hph gene, encoding hygromycin B phosphotransferase. We show that plasmid-borne hph can be expressed in fission yeast to confer hygromycin resistance. The vectors permit selection or screening in fission yeast for promoter activity of DNA fragments from other species. We used the vectors to identify several genomic sequences from Physarum that provide promoter function in fission yeast.

Cell-specific expression of a profilin gene family.

Profilin is a ubiquitous eukaryotic protein that inhibits actin polymerization. We cloned and sequenced the two profilin genes from the acellular slime mold Physarum polycephalum. The genes, proA and proP, each contain two introns. Primer extension experiments showed two possible transcription start sites in the profilin A gene and one start site in the profilin P gene. The profilin A mRNA has two polyadenylation sites, which yield mRNAs of about 600 and 500 nucleotides. The profilin P mRNA has a single polyadenylation site. The protein sequences were deduced from cDNA nucleotide sequencing. The profilin A and profilin P proteins contain 125 amino acids and are 66% identical in sequence. They show sequence similarity to Acanthamoeba (approximately 54%), yeast (approximately 46%), mouse (approximately 22%), calf (approximately 21%), and human (approximately 21%) profilins. The presence of the profilin A and profilin P mRNAs was studied throughout the life cycle. Profilin A mRNA was found in amebas, encysted amebas, and mature spores. The profilin P mRNA was present in plasmodia and spherulating plasmodia. Most cell types of P. polycephalum contain either the amebal or the plasmodial profilin mRNA, and no cell contains both mRNAs. This is the first evidence for developmental regulation of profilin isoforms.

Developmentally regulated late mRNAs in the encystment of Physarum polycephalum plasmodia.

Physarum polycephalum plasmodia survive adverse conditions by transforming into encysted cells called spherules. In this work we analysed the developmentally regulated mRNAs from the late stages of spherulation. A cDNA library was constructed and four abundant mRNAs were identified. One of the mRNAs was present in trace amounts in early spherules, while the other three were found only in late spherules. A cDNA clone for one of the late spherulation specific mRNAs was sequenced. It codes for a 332-amino-acid protein that did not show significant similarities with any known protein. Since the mRNA for this protein accumulates during spherulation, the protein was called spherulin 4. This protein has many features of a plasma membrane protein; it contains a signal peptide and a long hydrophobic region, which could serve as a transmembrane anchor. Another interesting feature is the presence of seven consecutive glycine residues in the N-terminal region. This is even more remarkable since the protein is not rich in glycine.