The nucleotide sequence of a developmentally regulated cDNA from Physarum polycephalum.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
D Pallotta
Publications and source records attributed to D Pallotta.
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.
Amino Acid Sequence↗
Replication timing of 10 developmentally regulated genes in Physarum polycephalum.
We have tested the hypothesis which stipulates that only early-replicating genes are capable of expression. Within one cell type of Physarum - the plasmodium - we defined the temporal order of replication of 10 genes which were known to be variably expressed in 4 different developmental stages of the Physarum life cycle. Southern analysis of density-labeled, bromodesoxyuridine-substituted DNA reveals that 4 genes presumably inactive within the plasmodium, were not restricted to any temporal compartment of S-phase: 1 is replicated in early S-phase, 2 in mid S-phase and 1 in late S-phase. On the other hand, 4 out of 6 active genes analysed are duplicated early, with the first 30% of the genome. Surprisingly, the two others active genes are replicated late in S-phase. By gene-dosage analysis, based on quantitation of hybridization signals from early and late replicating genes throughout S-phase, we could pinpoint the replication of one of these two genes at a stage where 80-85% of the genome has duplicated. Our results demonstrate that late replication during S-phase does not preclude gene activity.
Blotting, Southern↗
Expression of the three unlinked isocoding actin genes of Physarum polycephalum.
The actin gene family in Physarum polycephalum contains four unlinked loci: ardA, ardB, ardC, and ardD. The ardA locus is complex and probably contains two genes which we designated ardA2-7 and ardA2-17. cDNA clones corresponding to the ardB and ardC loci were isolated. Nucleic acid sequencing showed that these two cDNAs coded for the only abundant form of Physarum actin, which is 96% homologous to human gamma-cytoplasmic actin. The ardA2-17 gene also codes for this same actin protein (Nader et al., Gene 48, 133-144, 1986). The coding regions of ardB and ardC differ by 15 nucleotides. A comparison of the ardB and ardC sequences with ardA2-17 showed 73 and 77 nucleotide substitutions, respectively, in the coding regions. The noncoding regions of these three sequences were not homologous to each other or to the noncoding regions of actin genes from other organisms. Southern genomic hybridizations indicated that the ardA2-7 and ardD genes have weak sequence similarities to the three isocoding actin genes and thus form a different subclass of the family. Northern hybridizations showed that the ardB and ardC transcripts varied in abundance but were present in all the developmental stages. No ardA2-17 transcripts were seen. The relative abundance of the ardB and ardC transcripts was measured in amoebae and plasmodia by S1 nuclease protection and dot hybridization assays. A ratio of approximately 3:1 for ardC versus ardB was found for both stages. P. polycephalum is the first organism shown to contain three unlinked isocoding actin genes, of which at least two are expressed.
Actins↗
Selective gene expression during sporulation of Physarum polycephalum.
The two-dimensional gel electrophoresis of polypeptides synthesized in vitro from poly(A)+ RNA showed that mRNA populations change during sporulation of Physarum polycephalum. The differential hybridization of a cDNA library prepared from poly(A)+ RNA isolated from sporulating cells revealed that of 846 clones, 64 corresponded to sporulation-specific mRNAs. Further analysis demonstrated that these clones contained seven different sequences: three abundant sequences composing 3.2, 1.8, and 1.2% of the library and four other less abundant sequences. It is probable that all the major mRNAs specifically expressed in early stages of sporulation were identified. The most abundant mRNA from this group coded for a hydrophobic protein that contained a signal peptide. This protein is 47% similar to another Physarum protein, which was encoded by the most abundant plasmodium-specific mRNA. The plasmodial mRNA was degraded during sporulation and was replaced by the sporulation mRNA. These two proteins are thus encoded by members of a gene family whose expression is developmentally regulated.
Amino Acid Sequence↗
Gene families encode the major encystment-specific proteins of Physarum polycephalum plasmodia.
The encystment of Physarum polycephalum plasmodia, also called spherulation, involves the synthesis of many specific mRNAs and proteins. Most of these molecules accumulate at the onset of the major morphological and physiological changes typical of this differentiation pathway and are not present during the other two transitions leading to dormancy in Physarum, namely sporulation and encystment of amoebae. The nucleotide sequences of apparently full-length cDNA copies of the four major encystment-specific mRNAs were determined. The four sequences included the entire coding regions and at least 26 nucleotides of the 5'-nontranscribed leaders. The encoded proteins were named spherulins. We found that spherulins 1a and 1b are 81% homologous and are thus members of a gene family. They both possess putative signal peptides and N-glycosylation sites, suggesting that they are cell-wall glycoproteins. Spherulin 2a and spherulin 3a are non-homologous proteins. The absence of signal peptides suggests that they are intracellular structural proteins. Low-stringency Southern hybridizations showed that each also belongs to a two-member gene family.
Amino Acid Sequence↗
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.
Cloning, Molecular↗
Histone synthesis by lymphocytes in G0 and G1.
Peripheral blood lymphocytes are a naturally occurring population of G0 cells which can be activated in vitro to grow and divide. Upon activation with phytohemagglutinin (PHA), they enter G1 and, after a 24-h lag, begin DNA replication (S phase). Using radioisotope labeling and gel electrophoresis of acid-soluble chromatin proteins, we investigated histone synthesis in G0, G1, and S phase cultures of human and pig lymphocytes. In G0 and G1 cultures, which have less than 0.1% S phase cells, all five histones are synthesized and are incorporated into chromatin in equimolar amounts. In G0 lymphocytes histone synthesis accounts for at least 6% of nuclear protein radioactivity, and the rate of synthesis is about 2-3% of that of S phase lymphocytes. In contrast to histone synthesis by S phase cultures, G0 and G1 histone synthesis was completely resistant to treatment with hydroxyurea.
Animals↗
Changes in protein and RNA during asexual differentiation of Physarum polycephalum.
Some of the events during the growth and asexual differentiation of Physarum polycephalum amoebae are described. Encysted amoebae contain low levels of protein and RNA. When these cells are mixed with bacteria and inoculated onto agar plates, there is an increase in cellular RNA content followed by an increase in protein content. The cellular RNA and protein contents of all strains tested decrease during the subsequent cell divisions. In nondifferentiating cells (strain Cl at 30 degrees C and strain LU648), RNA and protein contents continue to decrease, and the cell eventually encyst. In the asexually differentiating strain Cl grown at 26 degrees C, the cellular RNA and protein contents stop decreasing and begin to increase when the first amoebae become committed to form plasmodia. At early stages of differentiation a new 36,000 molecular weight polypeptide appears. In fully formed plasmodia another polypeptide of 38,000 molecular is observed. These two plasmodial-specific polypeptides are among the most abundant plasmodial proteins.
Cell Division↗
Isolation and characterization of histones and other acid-soluble chromosomal proteins from Physarum polycephalum.
Chromosomal basic proteins were isolated from amoebal and plasmodial stages of the acellular slime mold Physarum polycephalum. Polyacrylamide electrophoresis on high resolution acid-urea gels separated the five histone fractions in the sequence H1, H2A, H2B, H3, and H4. Under these electrophoretic conditions Physarum histones migrated more like plant (rye) than animal (calf) histones. Furthermore, Physarum histones H1, H2A, and H2B have higher molecular weights on sodium dodecyl sulfate (SDS) gels than the corresponding calf fractions. No differences were detected between amoebal and plasmodial histones on either acid-urea or SDS-polyacrylamide gel electrophoresis. Amoebal basic proteins were fractionated by exclusion chromatography. The five histone fractions plus another major acid-soluble chromosomal protein (AS) were isolated. The Physarum core histones had amino acid compositions more closely resembling those of the calf core histones than of rye, yeast, or Dictyostelium. Although generally similar in composition to the plant and animal H1 histones, the Physarum H1 had a lower lysine content. The AS protein was extracted with 5% perchloric acid or 0.5 M NaCl, migrated between histones H3 and H4 on acid-urea polyacrylamide gels, and had an apparent molecular weight of 15 900 on SDS gels. It may be related to a protein migrating near H1. Both somewhat resembled the high mobility group proteins in amino acid composition.
Amino Acids↗
Yeast, rye, and calf histones. Similarities and differences detected by electrophoretic and immunological methods.
Yeast histones H2A, H2B, and H3 were purified using the standard histone purification procedures of differential solubility and exclusion chromatography. Yeast histone H4 was isolated by the same methods in a fraction containing one other major protein component. The four yeast core histones were identified by their reactions with antisera against rye and (or) calf histone fractions as well as by their electrophoretic, chromatographic, and solubility properties. The immunological distances between yeast H2B and rye and calf H2B fractions are substantial, as is the rye-calf distance for H2B. The immunological distance between yeast H2A and rye H2A is also large and is similar to the rye H2A - calf H2A distance. On the other hand, the immunological distance between yeast H3 and rye and calf H3 is much greater than that between rye H3 and calf H3. These and other results indicate that yeast H3 differs appreciably from the H3 of higher eucaryotes.
Animals↗
Conformations of calf thymus and rye histones H3 and H4 in aqueous solution by laser Raman spectroscopy.
The Raman spectra of aqueous solutions of histones H3 and H4 from calf thymus and from rye reflect the high degree of conservation from species to species of the primary and secondary structures of these proteins. The amount of beta-sheet structure is estimated at 40 +/- 5% in H4 and at 33 +/- 5% in H3 from the intensities of the amide I and amide III bands at 1663 and 1241 cm-1, respectively, in the spectra. These values are independent of the salt concentration of the solutions, mostly likely because of the high histone concentration (approximately 3 mM) required to obtain the spectra, which results in some aggregation of the proteins. The intensity ratio of the tyrosine doublet at 852 and 826 cm-1 indicates that the four tyrosine residues in H4 are relatively exposed to the solvent or weakly bound to positively charged groups of basic amino acids, whereas in H3 at least one tyrosine is buried inside the protein and tightly bound to a carboxylate group. The results also show that the secondary structure of H3 is slightly influenced by the state of oxidation of the two cysteine residues it contains.
Animals↗
Analysis of plant genomes. III. Denaturation and reassociation properties of cryptic satellite DNAs in barley (Hordeum vulgare) and wheat (Triticum aestivum).
A cryptic satellite fraction was isolated from barley and wheat by preparatory ultracentrifugation of total DNA in Ag+-Cs2SO4 density gradients and was characterized by studying its denaturation-reassociation properties. Wheat satellite DNA underwent thermal denaturation as a single component with a Tm of 81 degrees C while barley satellite DNA consisted of one major (Tm = 82.5 degrees C) and one minor (Tm = 91 degrees C) component. When the barley and wheat satellites were reassociated and then melted, the Tm values were found to be 6--7 degrees C lower than those of the corresponding native DNA preparations. Examination of the C0t curves of these two satellite DNAs revealed the presence of a major, fast reassociating and a minor, slow reassociating fraction. The fast reassociating DNA fraction of barley was found to have a complexity of 9.7 . 10(5) daltons while that of wheat satellite was 5.8 . 10(5) daltons. Since these satellites reassociated with about 4--5% base mismatching, as judged by their deltsTm (6--7 degrees C), they each appear to consist of rather similar base sequences.
Centrifugation, Density Gradient↗
Analysis of plant genomes. V. Comparative study of molecular properties of DNAs of seven Allium species.
The genomes of seven plant species belonging to the genus Allium and exhibiting a threefold variation in their nuclear DNA content were analyzed by studying their reassociation kinetics, equilibrium centrifugation behavior in neutral CsCl gradients, and melting properties. The reassociation kinetics experiments revealed the presence of 44-65% repeated DNA sequences. A comparison between DNA contents and the proportion of repeated DNA sequences indicated that, in Allium, increase in the genome size is not exclusively due to variations in the proportions of repetitive DNA. The total DNA as well as the various repetitive DNA fractions in all the Allium species examined exhibited, in spite of a few differences, a gross similarity in their behavior in neutral CsCl gradients and in their melting properties.
Base Sequence↗
Analysis of plant genomes. IV. Isolation and characterization of satellite DNA components from two dicotyledons cucumber (Cucumis sativus) and radish (Raphanus sativus).
Satellite DNA fractions from cucumber and radish, two plants having low DNA contents and relatively small chromosomes, were isolated and characterized. Reassociation studies of satellite and total nuclear DNA showed that the satellite fractions in these two plants contain most of the rapidly reassociating DNA. Cucumber satellite I was found to contain one major component (70% of the total satellite) having a density of 1.706 g/cm3 and a Tm of 90.5 degrees C and a minor component with a density of 1.712 g/cm3 and a Tm of 93.5 degrees C. The complexity of the major component was estimated to be 3.8 X 10(5) daltons while that of the minor one was 12.9 X 10(7) daltons. Although cucumber satellite II banded as a single peak at a density of 1.700 g/cm3 in neutral CsCl gradients, it was observed to have a rather broad denaturation profile with a Tm of 86.5 degrees C. Its Cot curve was also broader than that of satellite I and one of its components (40% of the total) had a complexity of 5.8 X 10(5) daltons. Two satellite fractions were also observed in the case of radish DNA but only satellite I was isolated in a pure form and characterized. This radish satellite formed a sharp, symmetrical peak at a density of 1.698 g/cm3 in neutral CsCl gradients and underwent denaturation in a narrow temperature range of 6 to 7 degrees C. An analysis of the optical reassociation kinetics showed that this satellite contained a major and a minor component. The major component, which comprised 80% of the satellite, had a complexity of 12.9 X 10(5) daltons. Hybridization experiments revealed that the ribosomal DNA was present in satellite II.
Centrifugation, Density Gradient↗
An immunological comparison of rye and calf histones.
The rye and calf histones were fractionated by differential solubility and exclusion chromatography. Antibodies were produced against histones H1, H2A, H2B, and H3 of both species and the extent of cross-reaction was measured by microcomplement fixation. The results allowed the identification of rye histones H2A and H2B. The near structural identity of the H3 histones was confirmed by the small immunological distance which indicated an amino acid difference of less than 2% between rye and calf. The interspecific differences found for both H2A and H2B were greater, the amino acid difference estimated from the immunological distance being between 10 and 20%. H1 gave no immunological cross-reaction, indicating a large (greater than 40%) difference in the structure of this protein in the two species. The extent of variation that a histone fraction shows between plant and animal species is probably related to its role in chromatin organization.
Animals↗
Laser Raman spectra of calf thymus histones H1, H2A, and H2B.
Laser Raman spectra of the calf thymus histones H1, H2A, and H2B in aqueous solutions are presented. The amide III band in the spectrum of the very lysine-rich histone H1 in aqueous solution appears at 1245 cm-1, which is almost at the same frequency as the corresponding vibration of the ionized form of poly(L-lysine). Upon increasing the NaCl concentration to 1 M, the frequency of the amide III vibration shifts to 1250 cm-1 as a result of the formation of a more compact disordered structure of at least the N-terminal region of the protein. Changing the pH from 3 to 5 induces the same frequency shift. The amide III regions of the Raman spectra of the slightly lysine-rich histones H2A and H2B shows two bands at 1247 and 1265 cm-1 for H2A, and at 1254 and 1265 cm-1 for H2B. These doublets are attributed to vibrations involving the backbone of at least two structurally distinct parts of the histone molecules. The low frequency component is assigned to the random-coil regions of the proteins which appear to have similar conformations for H1 and H2A. The frequency of this component also suggest that the structure of the disordered regions of H2B are more compact and less extended. These conclusions confirm the conformation predictions based on the primary structures of these proteins. The high frequency component at 1265 cm-1 is assigned to the alpha-helical and rigid disordered structures of H2A and H2B, since this band increases in intensity upon addition of NaCl. The amide I' region of the histone spectra is also presented but appears to be much less sensitive to the conformation than the amide III region. The intensity of the bands due to the single bond C-C stretching modes, as well as the intensity ratio of the tyrosine Fermi doublet at 855 and 830 cm-1, are also discussed.
Animals↗
Comparative study of rye and thymus histones: amino acid analysis and tryptic fingerprinting.
Amino acid composition and tryptic fingerprints of rye (Secal cereale) H1, H2B (PH1), and H2A(PHII) histones indicate the presence of major differences between these and the corresponding calf or rabbit fractions. In addition to variations for other amino acids, fraction H1 from rye contains twice as much arginine as the corresponding animal fraction; the plant H2B (PHI) and H2A (PHII) histones show lysine to arginine ratios greater than those of their animal counterparts. The tryptic maps of the same proteins appear to differ between plants and animals by the number and the general pattern of the peptides, as well as by the quantity and distribution of the arginine-containing peptides. Such results suggest the presence of differences in the primary structure of the calf and rye lysine-rich and moderately lysine-rich histones. Furthermore, the possibility is ruled out that each of these plant histones consists of an animal-like protein with an additional segment of 20--30 amino acid residues. On the other hand, the rye and calf arginine-rich fractions H3 and H4 show similar amino acid compositions and tryptic peptides maps.
Amino Acids↗