Dominick Pallotta

Electrophoretic study of plant histones: Comparison with vertebrate histones

Abstract The histones of seven plant species (barley, leek, onion, pea, radish, rye, and wheat) were isolated and compared to the histones of calf thymus and rat liver using electrophoresis on polyacrylamide-urea and polyacrylamide-SDS gels. It was found that the F1 histone of plants contains more subspecies and has generally higher molecular weights than their animal histone counterparts. Histones F3 of plants and animals have identical molecular weights and similar but not identical mobilities on polyacrylamide-urea gels. No histones were found in plants which have molecular weights and mobilities on polyacrylamide-urea gels which resemble the values for histones F2a2 and F2b of animals, but instead the series of histones observed differ from any of the animal histones. These plant histones may represent either substantially modified forms of F2a2 and F2b, or else may be a different class of histone molecules unique to plants. Fractions F2al in plants and animals are identical in electrophoretic behavior, but seem to differ in degree of acetylation.

Isolation and characterization of polymorphic microsatellite markers in the North Atlantic redfish (Teleostei: Scorpaenidae, genus Sebastes).

Redfish {genus Sebastes) are marine, viviparous and commercially important fish of the North Atlantic. For the last 30 years, a considerable amount of effort has been invested to study the evolutionnary relationships and the population genetic structure of tliis species complex. The complex is composed of four currently recognized taxa: Sebastes fasciatus, S. mentella, S. mariniis and S. vivipams. However, genetic studies relying on proteins and/or mitochondrial DNA markers have generally revealed low levels of genetic variation within and between redfish taxa (McGlade et al. 1983; Nedreaas & Naevdal 1991a), and hampered the analysis of young stages (Nedreaas & Naevdai 1991b). Consequently, there was a need for genetic markers with a higher resolution that could detect differences at any level of genetic differentiation (species, stocks), and any developmental stages. We report here the isolation and characterization of eight microsatelHtes from the Atlantic redfish complex that may be useful for a wide range of applications throughout the Atlantic Ocean.

Analysis of the genome of plants: II. Characterization of repetitive DNA in barley (Hordeum vulgare) and wheat (Triticum aestivum)☆

Barley and wheat DNAs have been characterized by studying their kinetics of reassociation, melting properties and sedimentation behaviour in neutral CsCl gradients as well as in Cs2SO4 gradients containing Ag+ or Hg2+. In both species, reassociation kinetics have revealed the presence of approx. 76% redundant nucleotide sequences which have been grouped into very rapidly reassociating (Cot 0-0.01), rapidly reassociating (Cot 0.01-1.0) and slowly reassociating (Cot 1-100) fractions. The barley Cot 0-0.01 and Cot 0.01-1.0 fractions as well as the wheat Cot 0.01-1.0 fraction form narrow bands upon centrifugation in CsCl gradients. Under similar experimental conditions both Cot 0.01 and Cot 1.0-100 wheat fractions and the barley Cot 1.0-100 fraction form broad bands each having several shoulders. Thermal denaturation studies of most of the above reassociated fractions have shown a considerable degree of order in their duplexes with an average hyperchromicity of 21.5%. When native, high molecular weight barley DNA is centrifuged in Ag+/CS2SO4 density gradients (RF = 0.2), two satellites appear on the heavier side of the main band, as against one in the case of wheat. The two minor peaks, designated as satellites I and II, have buoyant densities of 1.702 and 1.698 g/cm3, respectively, in neutral CsCl gradients and together represent about 8-9% of total barley DNA. Upon centrifugation in Hg2+/CS2SO4 density gradients, one satellite is observed in both barley and wheat and it accounts for 1-2% of their genomes.

Histones and RNA synthesis: Selective binding of histones by a synthetic polyanion in calf thymus nuclei

Abstract Polystyrene sulfonate, which decondenses facultative heterochromatin and activates RNA synthesis in male mealy bugs, and forms extremely tight complexes with histone, was added in increasing concentrations to calf thymus nuclei. The arginine rich fractions F3 and F2a1 are the first histone fractions to bind to Polystyrene sulfonate. When the weight of polystyrene sulfonate added is equal to the expected histone yield, all major histone fractions are bound to polystyrene sulfonate except the very lysine rich F1. A 10-fold increase in polystyrene sulfonate removes the F1. It is suggested that the removal of arginine rich histone at low polystyrene sulfonate concentration is related to activation of RNA synthesis in heterochromatin at low polystyrene sulfonate concentration, while the removal of very lysine rich histone at high polystyrene sulfonate concentration is related to the decondensation of chromatin at high polystyrene sulfonate concentration previously observed.

A gene,imz, affecting the pH sensitivity of zygote formation inPhysarum polycephalum

Mating inPhysarum polycephalum involves the fusion of two haploid amoebae and the differentiation of the resulting diploid zygote into a multinucleate plasmodium. Mating proceeds optimally with amoebae growing on an agar medium at pH 5.0. At pH 6.2, the amoebae still grow normally, but mating is completely blocked. The barrier at pH 6.2 is not in the differentiation step, since preformed diploids readily convert to plasmodia at this pH. The barrier can be overcome by raising the ionic strength of the agar medium; the effect, moreover, is not ion-specific. We have discovered a genetic locus,imz (ionicmodulation of zygote formation), that affects the upper pH limit for mating; the respective limits associated with the two known alleles,imz-1 andimz-2, are pH 5.6 and pH 6.0 at low ionic strength. Animz-1×imz-2 mating displays the pH 6.0 limit;imz-2 is therefore “dominant”. We suggest that this new gene affects a cell component that is exposed to the exterior of the amoeba and is involved in the fusion step of mating.

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.

Isolated histone fractions and the alkaline fast green reaction.

When the alkaline fast green reaction is applied to calf thymus histone fractions f1, f2a, f2b and f3 on Millipore filter paper it is found to stain equal amounts of these fractions equally well. The dye reaction, therefore, seems to depend on the over-all charge and not exclusively on the per cent arginine plus lysine in the histone fractions. This staining reaction has been applied to histone fractions separated by acrylamide gel disc electrophoresis. Deamination of the histone fractions on both paper and acrylamide gel resulted in a reduction in the intensity of the stain. The stain that remained followed the relative amount of arginine in each fraction or band. In the gels, the f1 band was removed completely. This confirms previous interpretations of the cytochemical deamination reaction. The stain reactions have been utilized as a means of comparing electrophoretically resolved histone bands of similar mobility from crickets and mealy bugs.

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.

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.

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 pfy1Δ mutant. Electron microscopic observations reveal an accumulation of glycoproteins in the bud and in the mother cell. The MATa, pfy1Δ cells mate as well as wild‐type cells, while the mating efficiency of MATα, pfy1Δ cells is reduced. Pulse‐chase experiments demonstrate an accumulation of the 19 kDa α‐factor precursor and delayed secretion of the mature α‐factor. The TGN protein Kex2p is the principal enzyme responsible for the endoproteolytic cleavage of the α‐factor precursor. An immunofluorescence detection of Kex2p shows an altered localization in pfy1Δ 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, α‐factor maturation and the accumulation of intracellular membranous structures in pfy1Δ cells.