Counteraction of NaCl with NaH2PO4 and NaNO3 on pigment,saccharide and protein contents in broad bean

Salinity inhibited growth, and affected the contents of chlorophylls, carotenoids, saccharides, amino acids, proteins, DNA and RNA in broad bean plants. Foliar application of NaH₂PO₄ and NaNO₃ greatly ameliorated the adverse effects of NaCI. This counteraction was associated with an increase in contents of saccharides, proteins, DNA and RNA.     

Changes in contents of chlorophyll,proteins, and saccharides in leaves during plant development of determinate,semideterminate, and indeterminate lines of soybean

Major differences betwean the determinate (dt₁Dt₂,dt₁dt₂), semideterminate (Dt₁Dt₂), and indeterminate (Dt₁dt₂) near-isogenic lines of glycine max (L.) Merr, mainly appeared after R1 (reproductive) stage. Increases in specific leaf matter (SLM) between Rl and R6 stages showed that determinate lines have higher SLM than semideterminate or indeterminate lines. Soluble protein and starch also accumulated more rapidly in determinate lines. Insoluble protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA), reducing saccharides, chlorophyll, and soluble saccharides contents of leaf from determinate lines resembled those of semideterminate and indeterminate lines.     

Alleviation of the adverse effects of NaCl on germination of maize grains by calcium

The lengths of roots and shoots, fresh and dry matter yield, and the contents of insoluble saccharides and free amino acids were reduced with the rise in NaCl concentration. However, under combination of NaCl with Ca²⁺ ions, these parameters generally raised. Contents of soluble saccharides, proline and quaternary ammonium compounds increased with increasing NaCl concentration, but under addition of CaCl₂ or CaSO₄, contents of these compounds were decreased. Low concentrations of NaCl stimulated soluble proteins, production, but higher concentrations decreased the content of soluble proteins. Addition of Ca²⁺ in the media did not improve the soluble protein production. Insoluble proteins content was increased with the rise of salinity level, but these effects were more pronounced with NaCl and CaCl₂ or CaSO₄ than with NaCl only.     

Site-specific cleavage of DNA–RNA hybrids by zinc finger/FokI cleavage domain fusions

Zinc-finger proteins of the Cys₂His₂ type bind DNA–RNA hybrids with affinities comparable to those for DNA duplexes. Such zinc-finger proteins were converted into site-specific cleaving enzymes by fusing them to the FokI cleavage domain. The fusion proteins are active and under optimal conditions cleave DNA duplexes in a sequence-specific manner. These fusions also exhibit site-specific cleavage of the DNA strand within DNA–RNA hybrids albeit at a lower efficiency (≃50-fold) compared to the cleavage of the DNA duplexes. These engineered endonucleases represent the first of their kind in terms of their DNA–RNA cleavage properties, and they may have important biological applications.     

Growth and some metabolic activities ofScenedesmus obliquus cultivated under different NaCl concentrations

The physiological response ofScenedesmus obliquus to salinity (NaCl concentration of 40, 80, 120, 60 and 200 mM) for 7 d (long-term experiments) or 2 h (short-term experiments) was followed. Cell number, dry matter and the content of photosynthetic pigments decreased with the rise of NaCl concentration. However, the photosynthetic O₂ evolution mostly increased with the increase of NaCl concentration up to 80 mM, and respiration (dark O₂ uptake) was markedly promoted. Photosynthesis/respiration ratio went in concomitance with the cell number, dry matter or chlorophyll content. Contents of soluble saccharides and soluble proteins increased with the rise of salinization, while the content of insoluble and total saccharides or proteins decreased. Proline content increased greatly with salinization, whereas of other free amino acids were mostly reduced, especially at higher salinities. Similarly, the lipoid contents of salinizedScenedesmus obliquus were obviously higher than those of the control cultures.     

Effects of Phytoplasma Infection on Growth and Photosynthesis in Leaves of Field Grown apple (Malus Pumila Mill. cv. Golden Delicious)

The contents of chlorophyll (Chl), leaf biomass, and soluble proteins were markedly decreased in phytoplasma infected apple leaves. Similar results were also observed for ribulose-1,5-bisphosphate carboxylase, ¹⁴CO₂ fixation, and nitrate reductase activity. In contrast, the contents of sugars, starch, amino acids, and total saccharides were significantly increased in phytoplasma infected leaves. In isolated chloroplasts, phytoplasma infection caused marked inhibition of whole photosynthetic electron chain and photosystem 2 (PS2) activity. The artificial exogenous electron donor, diphenyl carbazide, significantly restored the loss of PS2 activity in infected leaves. Similar results were obtained when F_v/F_m was evaluated by in vivo Chl a fluorescence kinetic measurements.     

The ultrasensitive silver "protein" stain also detects nanograms of nucleic acids

The ultrasensitive silver staining procedure developed for proteins also stains nanogram quantities of RNA and DNA in polyacrylamide gels. A gradient polyacrylamide gel system is described which separates proteins from 10⁴ to 10⁶ M_r, RNA from 5S to 23S and DNA from 0.4 to 21 Kb. The sensitivity of nucleic acid silver staining in this gel system considerably exceeds that of commonly used DNA and RNA dye-binding stains.     

Tsetse Salivary Gland Proteins 1 and 2 Are High Affinity Nucleic Acid Binding Proteins with Residual Nuclease Activity

Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans) saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2) display DNA/RNA non-specific, high affinity nucleic acid binding with K_D values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.     

Interphase chromosomal deoxyribonucleoprotein isolated as a discrete structure from cultured cells

A new procedure is described for the preparation of interphase chromatin from cultured mouse cells (line P815). The primary objective of this procedure was to eliminate exchanges of histones between deoxynucleoprotein molecules; this objective is shown experimentally to have been attained. The chromatin is released from cells by the non-ionic detergent Nonidet P40 in medium of low ionic strength (0.1 mM-KNa₂PO₄), and may then be sedimented as a structure which conserves the general form and ultrastructural characteristics of chromatin within the cell. The nuclear envelope cannot be detected in these structures by electron microscopy, and their content of choline-containing phospholipids is less than 10% of that of nuclei. The maintenance of form in this structure must thus depend on properties of the chromatin itself, and possibly on the more compact peripheral chromatin.Soluble DNP² prepared by shearing these structures has the same relative contents of DNA, histones, non-histone proteins and RNA as DNP prepared by standard methods. Analyses by electrophoresis on polyacrylamide gels of the non-histone proteins reveals certain differences from the pattern of these proteins in DNP prepared by a salt precipitation method. The template activity for RNA synthesis, in the presence of Escherichia coli RNA polymerase of sheared, soluble DNP prepared by this procedure, is comparable to that of DNP prepared by other methods. However, in the absence of exogenous RNA polymerase the rate of RNA synthesis by structured (unsheared) chromatin is about ten times higher than the rate using sheared DNP.The rapid removal of the nuclear envelope in this lysis procedure allowed experimental examination of the origin of the histones and non-histone proteins of DNP. When DNP was prepared from a mixture of two populations of cells, one containing DNA distinguishable by a density label and the other containing radioactively labelled proteins, radioactive proteins were found exclusively in DNP of normal density, and not in dense DNP and vice versa. It is concluded that the proteins of DNP prepared in this way are not acquired during the preparation procedure but were already associated with DNA in vivo, and that other proteins are not bound non-specifically to DNA during the preparation of DNP. When a mixture of DNP molecules prepared, in this way is precipitated in 150 mm-NaCl and redissolved, some radioactively labelled histones migrate onto dense DNA molecules.This procedure is suitable for routine, quantitative isolation of chromosomal DNP from small numbers of cells; it is also applicable to cells of other cultured lines.     

DNA,RNA, and protein synthesis in the cyanobacteriumSynechocystis sp. PCC 6803 adapted to different salt concentrations

A salt shock of 684mm NaCl reduced RNA and DNA synthesis to about 30% of the control level inSynechocystis. DNA synthesis recovered to the initial level within 4 h, while for recovery of RNA synthesis about 8 h were necessary. In cells completely adapted to different salt concentrations (from 171 to 1026mm NaCl), a continuous decrease in the RNA content with increasing salt concentrations up to 684mm NaCl was found, whereas the lowest DNA content was measured around 342mm NaCl, i.e., the salinity at which maximal growth occurred. With the uracil and thymidien incorporation technique, maxima in DNA and RNA synthesis were detected in control cells. Comparing these rates with nucleic acid synthesis rates calculated from the contents of DNA and RNA and the growth rates indicated that adaptation to 1026mm NaCl seemed to lead to an increased RNA turnover inSynechocystis. Analysis of protein synthesis with³⁵S-methionine labeling showed alterations in salt-adapated cells ofSynechocystis. At least three proteins (20.5, 25.8, and 35.8 kDa) were synthesized with highest rates at salinities leading to maximal growth, the synthesis of nine proteins (12.5, 16.9, 19.2, 22.2, 24.7, 28.5, 30.5, 50.3, and 63.5 kDa) increased and that of several other proteins decreased with increasing salinity; but only three proteins (12.5, 22.2, and 30.5 kDa) accumulated under these conditions. The adaptation ofSynechocystis to enhanced salt concentrations led also to increased contents of glucosylglycerol, glycogen, and significant amounts of K⁺ as well as Na⁺ ions.     

Cuprolinic Blue: a specific dye for single-stranded RNA in the presence of magnesium chloride. I. Fundamental aspects

Summary Qualitative and quantitative aspects of the cationic dye Cuprolinic Blue were investigated with model films of polyacrylamide gel in which RNA, DNA and other biological polyanionic compounds had been incorporated. In the presence of 1m MgCl₂, Curpolinic Blue was found to bind specifically to single-stranded RNA, leaving native DNA, proteins, (acid) polysaccharides and phospholipids completely unstained. Under these conditions, Cuprolinic Blue is complexed by non-electrostatic bonds with non-stacked purine bases, mainly adenine. Optimal conditions for dye binding and differentiation have been defined. Both the Cuprolinic Blue-MgCl₂ staining of single-stranded RNA and the Cuprolinic Blue staining of RNA and DNA in the absence of MgCl₂ were found to obey the Lambert-Beer law. The advantages and possible applications of Cuprolinic Blue are compared with well-known (indirect) histochemical RNA staining procedures.     

Basal-Cell Subpopulations and Cell-Cycle Kinetics In Human Epidermal Explant Cultures

Cultured human epidermal cells were studied by cell sorting and autoradiography after different ³H-thymidine (³H-dThd)-labelling procedures and after labelling with DNA precursors that are incorporated via salvage or de novo pathways. It was shown that ³H-dThd incorporation was the best measure of the rate of DNA replication. Dose-response experiments with pulse and continuous labelling revealed that all S- and G₂-phase cells were cycling, whereas some 20% of the cells stayed in G₁-phase for long periods of time. Most, if not all of these cells were probably non-proliferating differentiated keratinocytes. At least two subpopulations of S-phase cells could be discriminated on the basis of the rate of incorporation of DNA precursors. the difference in precursor incorporation did not seem to be caused by differences in nucleotide metabolism but rather to reflect true differences in the rate of DNA replication. Continuous labelling experiments showed that these subpopulations also were apparent in the G₁- and G₂-phases. Studies of the grain-count distribution revealed that cells that appeared to move rapidly through the S-phase moved slowly through the G₂-phase, and vice versa. Cells stained with acridine orange were subjected to a two-parameter analysis in the cell sorter by simultaneous measurement of the DNA and RNA fluorescence. Autoradiography of sorted cells revealed that, on average, cells with low RNA contents incorporated ³H-dThd at a higher rate than cells with high RNA contents.     

Intranucleolar visualization of nucleic acids and acidic proteins in inhibited and reactivated pea cotyledonary buds

Summary Nuclease-colloidal gold complexes and silver staining were used to visualize intranucleolar nucleic acids and argyrophilic proteins of the nucleolar organizers in bud cotyledonary cells ofPisum sativum. In the G_0–1 inhibited bud, a few RNA molecules were detected in the fibrillar component and in the unique fibrillar centre, close to the boundary with the fibrillar component of the nucleolus. DNA was present in the fibrillar component, in the fibrillar centre and in a few fibres crossing the perinucleolar halo. The acidic proteins were localized at the periphery of the fibrillar component but they were also present in the unique fibrillar centre. In the reactivated bud, RNA was particularly concentrated in the granular component and along fibres crossing the perinucleolar halo; a few RNA molecules were also detected at the boundary between the small fibrillar centres and the fibrillar component. DNA was localized in the same nucleolar component as in the inhibited bud, but it was distributed between several fibrillar centres. Acidic proteins coated these DNA loci. In the inhibited and reactivated bud connections between nucleolar DNA containing structures were displayed. The data are discussed in relation to the present knowledge of the functional architecture of the nucleolus.Abbreviations DNA deoxyribonucleic acid - DNase deoxyribonuclease - G_0–1 phase G₁ phase of the cell cycle indefinitely prolonged - PEG polyethylene glycol - RNA ribonucleic acid - RNase ribonuclease - S and G₂ phases synthetic and postsynthetic phases of the cell cycle - SPB saline phosphate buffer     

Replication initiated at the origin (oriC) of the E. coli chromosome reconstituted with purified enzymes

A crude soluble enzyme system capable of authentic replication of a variety of oriC plasmids has been replaced by purified proteins constituting three functional classes: initiation proteins (RNA polymerase, dnaA protein, gyrase) that recognize the oriC sequence and presumably prime the leading strand of the replication fork; replication proteins (DNA polymerase III holoenzyme, single-strand binding protein, primosomal proteins) that sustain progress of the replication fork; and specificity proteins (topoisomerase I, RNAase H₁ protein HU) that suppress initiation of replication at sequences other than oriC, coated with dnaA protein. Protein HU and unidentified factors in crude enzyme fractions stimulate replication at one or more stages. Replication has been separated temporally and physically into successive stages of RNA synthesis and DNA synthesis.     

Polyamines and senescence of maintenance foliage of tea,Camellia sinensis L.

In leaf discs of maintenance foliage of tea (Camellia sinensis) polyamines (PA_s) and kinetin retarded chlorophyll (Chl) loss, whereas inhibitors of PA biosynthesis [difluoromethyl arginine, difluoromethyl ornithine, methylglyoxal-bis(guanylhydrazone)] and abscisic acid (ABA) promoted senescence. The contents of RNA and protein were significantly higher in PA and kinetin treated leaf discs as compared to those treated with inhibitors and ABA. The contents of total and reducing saccharides declined with the progressive loss of Chl, and the concentration of starch increased in all the PA treated leaf discs. Free amino acid content also increased under all the treatments, but the increase was comparatively larger in case of inhibitors application. The authors thank Director, CSIR Complex, Palampur for providing necessary facilities and Merrel Dow Research Institute (Cincinnati, OH) for generous gift of DFMO and DFMA.     

“Protein aggregates” contain RNA and DNA,entrapped by misfolded proteins but largely rescued by slowing translational elongation

All neurodegenerative diseases feature aggregates, which usually contain disease‐specific diagnostic proteins; non‐protein constituents, however, have rarely been explored. Aggregates from SY5Y‐APP_Sw neuroblastoma, a cell model of familial Alzheimer''s disease, were crosslinked and sequences of linked peptides identified. We constructed a normalized “contactome” comprising 11 subnetworks, centered on 24 high‐connectivity hubs. Remarkably, all 24 are nucleic acid‐binding proteins. This led us to isolate and sequence RNA and DNA from Alzheimer''s and control aggregates. RNA fragments were mapped to the human genome by RNA‐seq and DNA by ChIP‐seq. Nearly all aggregate RNA sequences mapped to specific genes, whereas DNA fragments were predominantly intergenic. These nucleic acid mappings are all significantly nonrandom, making an artifactual origin extremely unlikely. RNA (mostly cytoplasmic) exceeded DNA (chiefly nuclear) by twofold to fivefold. RNA fragments recovered from AD tissue were ~1.5‐to 2.5‐fold more abundant than those recovered from control tissue, similar to the increase in protein. Aggregate abundances of specific RNA sequences were strikingly differential between cultured SY5Y‐APP_Sw glioblastoma cells expressing APOE3 vs. APOE4, consistent with APOE4 competition for E‐box/CLEAR motifs. We identified many G‐quadruplex and viral sequences within RNA and DNA of aggregates, suggesting that sequestration of viral genomes may have driven the evolution of disordered nucleic acid‐binding proteins. After RNA‐interference knockdown of the translational‐procession factor EEF2 to suppress translation in SY5Y‐APP_Sw cells, the RNA content of aggregates declined by >90%, while reducing protein content by only 30% and altering DNA content by ≤10%. This implies that cotranslational misfolding of nascent proteins may ensnare polysomes into aggregates, accounting for most of their RNA content.     

Effect of vitamin B6 deficiency on pancreatic acpinar cell function

The present study was designed to determine the effect of vitamin B₆ deficiency on pancreatic acinar cell function. Rats were either fed $\text{ad}$$\text{lib}$ or rendered B₆-deficient by a purified B₆-deficient diet; half of the latter being replenished with IP pyridoxine before sacrifice. Body weight, pancreatic weight, RNA and DNA content were decreased in B₆-deficient animals. These changes were considered to be due to inanition resulting from decreased food intake. Amylase content of pancreas in B₆-deficient animals was less compared with B₆-replenished animals. Although slightly higher in B₆-deficient animals, the incorporation of L-phenylalanine¹⁴C into total tissue proteins was not significantly different in the three groups of animals. On B₆-replenishment, incorporation of L-phenylalanine¹⁴C into nascent proteins was diminished in spite of higher tissue amylase and protein content. Vitamin B₆ deficiency decreased total RNA content and adenine-8-¹⁴C incorporation into RNA. DNA content was diminished but incorporation of thymidine-2-¹⁴C into DNA was increased. On replenishment with B₆, thymidine-2-¹⁴C incorporation decreased significantly compared to control animals. Secretion of amylase was diminished commensurate with decreased content. It is concluded from these studies that B₆-deficiency induced DNA injury, decreased RNA turnover and increased protein turnover resulting in diminished amylase content. These data indicate that B₆-deficiency so frequently encountered in alcoholism may contribute to the pancreatic injury in this clinical condition.     

Contractile roots are the most sensitive organ in <Emphasis Type="Italic">Crocus sativus</Emphasis> to salt stress

Crocus sativus corms were grown in Perlite and watered by half-strength modified Hoagland nutrient solution containing 0, 50, 100, 150, 200 mM NaCl. Growth parameters and contents of proteins, proline, polyphenols, minerals and saccharides were studied in fibrous roots, contractile roots, corms and leaves. All plants remained alive and did not display any sign of foliar damage even at 200 mM NaCl. However, the salinity decreased growth, relative water content and increased contents of proline and Na⁺ in all organs. Total protein content was increased in corms and contractile roots but decreased in fibrous roots. Changes in protein pattern were also observed. Polyphenol content was increased by salinity in all organs except the leaves. As salinity increased, content of soluble saccharides decreased except in the contractile roots.