Gérald Lemieux

Molecular cloning of mRNAs expressed specifically during spherulation of Physarum polycephalum

A cDNA library was constructed using the poly(A)+ RNA extracted from spherulating Physarum polycephalum microplasmodia. This library (740 clones) was screened by differential hybridization with 32P-labeled poly(A)+ RNA from growing plasmodia and developing spherules. The results showed that at least 30% of the clones corresponded to mRNAs expressed specifically in spherulating plasmodia. The 35 spherulation-specific cDNA clones giving the strongest hybridization signals were analysed. From this group, four different sequences complementary to very abundant mRNAs were identified. They each accounted for 1.5% of 4.5% of all the clones in the library and probably represented the most abundant spherulation-specific mRNAs. In addition, four less abundant mRNAs were identified from stage-specific clones giving weaker hybridization signals. These sequences represented individually between 0.3% and 0.7% of the clones in the library. Northern blots showed that these eight different sequences were absent from plasmodia and were most abundant 24-36 h after the induction of spherulation. Similar results were also obtained when spherulation was induced by the addition of a sublethal concentration of ferrous iron ions to the growth medium. Hybridization of the spherule-specific clones to Southern blots of genomic DNA suggested the presence of one copy for each gene.

Purification and characterization of plasma membranes from Physarum polycephalum amoebae

Abstract In this study we describe a method for preparing plasma membranes from Physarum polycephalum amoebae. The cells were swollen in hypotonic medium (1 mM ZnCl 2 , 10 mM Tris-HCl, pH 8.0) and then broken in a Thomas tissue grinder fitted with a teflon pestle. The plasma membranes were collected by differential centrifugation and purified by centrifugation on a continuous 20–50% sucrose gradient. The membranes sedimented in a single band having a density of 1.16 g/cm 3 . They were found by enzymatic assay and by electron microscopy to be free of lysosomes, mitochondria, and nuclei and minimally contaminated by endoplasmic reticulum. The membrane proteins were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis; 10 major and 20 minor bands were seen. Periodic acid Schiffs staining of electrophoretically separated proteins revealed five major and six minor bands containing glycoproteins. Radioiodinated cell surface proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis into six bands with apparent weights greater than 68 000.

Ribosomal phosphoproteins in Physarumpolycephalum

Abstract Ribosomal proteins of Physarum polycephalum were labelled in vivo with 32PO4. Three acid phosphoproteins were observed in the large subunit, while two basic ones were present in the small subunit. Ribosomal phosphoprotein S3 accounted for 70% of the total radioactivity and may be equivalent to S6 from rat liver.

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.

Different developmental programs for amoebal and plasmodial encystment in Physarum polycephalum

SummaryAmoebal and plasmodial encystment (spherulation) of Physarum polycephalum were compared. Encystment of amoebae was induced by the addition of glucose to a dense culture. A change in cellular proteins during the amoeba to rnicrocyst transition was demonstrated by SDS-polyacrylamide gel electrophoresis. We also observed that the encystment of amoebae did not involve the accumulation of the proteins which accompany plasmodial encystment. A cDNA library was constructed from poly(A)+ RNA of encysted amoebae and was screened by differential hybridization. Two different mRNAs, specific to mature microcysts were identified. These mRNAs were not found in encysted plasmodia. Likewise, four encysted plasmodial mRNAs were absent from microcysts. These results show that different developmental programs are used for amoebal and plasmodial encystment.

cDNA Cloning Of Physarum polycephalum Stage-Specific mRNAs

A useful approach to the study of differentiation consists of isolating and characterizing the genes specifically expressed in the various stages and then studying their regulation. This chapter describes the methodology used in our laboratory to clone sequences from stage-specific mRNAs of Physarum polycephalum. It involved the preparation of intact poly (A)+ RNA and the construction of cDNA libraries for four developmental stages (amoebae, plasmodia, spores, and spherules). These libraries were then screened by differential hybridization, and the specificity of the positive clones was confirmed by Northern blot hybridization. The quality of the results obtained at each step was controlled, and the methodology was found to be of general use in the identification of Physarum stage-specific mRNAs.

Phosphorylation of ribosomal proteins S3, L1 and L24 during spherulation in Physarum polycephalum

The phosphate content of ribosomal proteins S3, L1 and L24 has been determined in the course of spherulation of Physarum polycephalum. The major phosphoprotein, S3, was completely dephosphorylated after 4 h of differentiation. The phosphate content of L1 and L24 was not altered during the differentiation. The cellular level of ATP remained constant for at least 5 h. A 3-fold reduction of cyclic AMP concentration occurred in the first hour, followed by a slow increase to a final value of twice the level observed in growing cells. The results showed that the phosphorylation of ribosomal proteins is regulated by at least two different mechanisms and that the dephosphorylation of S3 is not induced by a lack of cellular ATP. Although cyclic AMP might trigger the dephosphorylation of S3, the phosphate content of this protein remained at a very low value even when the cellular concentration of cyclic AMP rose significantly. Since the polysome level remains constant during the first 24 h of spherulation, the phosphorylation of S3 is not necessary for active protein synthesis and the phosphorylation of L1 and L24 is not involved in ribosome inactivation, which occurs after 24 h.

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.

Ribosomes of Physarum polycephalum. Subunits and protein composition.

Ribosomes from Physarum polycephalum were purified. Optimal conditions for preparation and stability of subunits were determined. KCl concentration above 200 mM induced protein dissociation from the subunits. It was observed that dissociated ribosomes were more stable in a low ionic strength buffer than in 200 mM KCl, where the 40 S was preferentially degraded by ribonucleases. Ribosomal proteins were analyzed by two-dimensional gel electrophoresis. The first dimension was carried out at pH 8.6 while the second was run at pH 4.6. The monosome contained sixty seven proteins, of which six were acidic. Two proteins were lost after subunit dissociation. Twenty six basic and two acidic proteins were observed in the 40 S subunit while the largest subunit gave thirty nine spots on the basic part of the gel and three additional spots on the acidic side. Five proteins were shared by 40 S and 60 S.

Phosphorylation of ribosomal proteins during the cell cycle of Physarum polycephalum

Physarum polycephalum has been used as a model system to study the phosphorylation of ribosomal proteins during the cell cycle. The results showed that the phosphate content of S3, the major ribosomal phosphoprotein in this organism, was constant during all phases of the cell cycle. No additional ribosomal phosphoproteins were observed. These results differ significantly from those reported earlier by Rupp, R.G., Humphrey, R.M. and Shaeffer, J.R. (Biochim. Biophys. Acta (1976) 418, 81-92) and suggest that the use of thymidine or hydroxyurea to synchronize cell population may affect the phosphorylation of ribosomal proteins. The results are discussed in relation to protein synthesis and cAMP level during the cell cycle.