Protein variation accompanies leaf dedifferentiation in sugarcane (Sacchamm officinarum) and is influenced by genotype.

Tissue culture lines (morphogenic and nonmorphogenic) were established in five genotypes of Saccharum officinarum L. Changes in protein expression after dedifferentiation of leaf tissue into callus were investigated by two-dimensional gel electrophoresis of cellular proteins. The findings demonstrated that protein expression was regulated both quantitatively and qualitatively in all five genotypes of sugarcane. Sixty-three dedifferention-proteins were identified, of which thirty-three were common to all genotypes. The expression of the remainder was dependent on the genotype, suggesting that the genotype within the same sugarcane species plays an important role in dedifferentiation. Three of the dedifferentiation-proteins were specific to morphogenic callus lines and one to nonmorphogenic callus lines. These proteins can be useful in characterizing the biochemical, molecular, and genetic properties of cultured cells in sugarcane, and in understanding the influence of genotype in the induction of dedifferentiation-proteins and their role in morphogenesis.

The Dictyostelium ribosome: biochemistry, molecular biology, and developmental regulation.

This is the first comprehensive review of ribosomes in the cellular slime mold Dictyostelium discoideum. The physicochemical, biochemical, cellular, molecular, and developmental properties are reviewed. Several features demonstrate that a unique class of ribosomes exists in this organism, and a study of these ribosomes will be important to decipher special features of translational regulation, and evolution of the organelle in the eukaryotic kingdom.

Differences in accumulation and phosphorylation of proteins in vegetatively growing and aggregation-competent cells of Dictyostelium discoideum.

A comparison of proteins from whole cell lysates of vegetative amoebae and aggregation-competent cells by high-resolution two-dimensional gel electrophoresis coupled with a sensitive silver staining method revealed distinct differences. In aggregation-competent cells, 16 proteins present in the vegetative amoebae disappeared, and 25 new proteins appeared. A few other proteins showed quantitative variation during the transition of vegetative amoebae to aggregation competence. Identification of phosphoproteins by in vivo labeling with [32P]orthophosphate showed that none of the developmentally regulated cellular proteins were modified. Phosphorylation was observed in four proteins. One protein was phosphorylated exclusively in aggregation-competent cells. The phosphorylation level of two other proteins was higher in aggregation-competent cells compared with vegetative amoebae. The data suggest that phosphorylation of cellular and certain ribosomal proteins may be regulated coordinately in Dictyostelium discoideum.

Covalent modifications of ribosomal proteins in growing and aggregation-competent dictyostelium discoideum: phosphorylation and methylation.

Phosphorylated and methylated ribosomal proteins were identified in vegetatively growing amoebae and in the starvation-induced, aggregation-competent cells of Dictyostelium discoideum. Of the 15 developmentally regulated cell-specific ribosomal proteins reported earlier, protein A and the acidic proteins A1, A2, and A3 were identified as phosphoproteins, and S5, S6, S10, and D were identified as methylated proteins. Three other ribosomal proteins were phosphorylated and 19 others methylated. S19, L13, A1, A2, and A3 were the predominant phosphoproteins in growing amoebae, whereas S20 and A were the predominant ones in the aggregation-competent cells. Among the methylated proteins, eight (S6, S10, S13, S30, D, L1, L2, and L31) were modified only during growth phase, six (S5, S7, S8, S24, S31, and L36) were altered only during aggregation-competent phase, and nine (S9, S27, S28, S29, S34, L7, L35, L41, and L42) were modified under both phases. Five proteins (S6, S24, L7, L41, and L42) were heavily methylated and of these, the large subunit proteins were present in both growing amoebae and aggregation-competent cells. These findings demonstrate that covalent modification of specific ribosomal proteins is regulated during cell differentiation in D. discoideum.

Induction of cell-specific ribosomal proteins in aggregation-competent nonmorphogenetic Dictyostelium discoideum.

Vegetatively growing amoebae, if shaken in a starvation (nonnutrient) buffer, acquired aggregation competence, but do not embark on a morphogenetic program. The quantitative variation of ribosomal proteins in vegetative and aggregation-competent cells was compared by labeling the different cell types with [35S]methionine. Vegetative cells were examined at various phases of the growth cycle. No changes could be detected in the content of ribosomes or the apparent stoichiometry of ribosomal proteins in growing cells. In stationary phase cells, the net ribosome content declined to 15% of that observed in logarithmic phase, but the relative amounts of individual ribosomal proteins were not altered. Although aggregation-competent cells contained 30% less ribosomes compared with logarithmic phase cells, the total fraction of newly made ribosomal proteins was the same in both. In contrast to vegetative cells, distinct changes were induced in the ribosomal proteins of aggregation-competent cells. The composition of ribosomes in aggregation-competent phase resembled in every respect that observed in spore cells. As reported earlier, changes were found in all 12 of the developmentally regulated ribosomal proteins. For the majority of newly made ribosomal proteins during aggregation competence, the stoichiometry was similar to that in logarithmically growing cells. However, the relative synthesis of some was particularly higher (13- to 46-fold for A and L; 3- to 8-fold for D, E, S24, L3, S6, and L4) compared with logarithmic phase cells. About 18 proteins, which included the cell-specific ribosomal proteins L18, S10, S14, S16, and L11, were synthesized in lesser amounts than in logarithmic phase cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Subcellular distribution of ribosomal proteins in Dictyostelium discoideum.

The distribution of ribosomal proteins in monosomes, polysomes, the postribosomal cytosol, and the nucleus was determined during steady-state growth in vegetative amoebae. A partitioning of previously reported cell-specific ribosomal proteins between monosomes and polysomes was observed. L18, one of the two unique proteins in amoeba ribosomes, was distributed equally among monosomes and polysomes. However S5, the other unique protein, was abundant in monosomes but barely visible in polysomes. Of the developmentally regulated proteins, D and S6 were detectable only in polysomes and S14 was more abundant in monosomes. The cytosol revealed no ribosomal proteins. On staining of the nuclear proteins with Coomassie blue, about 18, 7 from 40S subunit and 11 from 60S subunit, were identified as ribosomal proteins. By in vivo labeling of the proteins with [35S]methionine, 24 of the 34 small subunit proteins and 33 of the 42 large subunit proteins were localized in the nucleus. For the majority of the ribosomal proteins, the apparent relative stoichiometry was similar in nuclear preribosomal particles and in cytoplasmic ribosomes. However, in preribosomal particles the relative amount of four proteins (S11, S30, L7, and L10) was two- to four-fold higher and of eight proteins (S14, S15, S20, S34, L12, L27, L34, and L42) was two-to four-fold lower than that of cytoplasmic ribosomes.

Protein polymorphism in sugarcane revealed by two-dimensional gel analysis.

In order to identify molecular markers for the analysis of the sugarcane genome, proteins extracted from apical segments of shoot tissues were resolved by a combination of equilibrium (IEF) and nonequilibrium (NEPHGE) two-dimensional polyacrylamide gel electrophoresis. A number of taxa of the "Saccharum complex" group (Saccharum species and the related genera of Andropogoneae) with presumed contributions to the sugarcane genome were surveyed. Protein profiles were compared to a reference map consisting of 1,482 protein spots from the noble cane,Saccharum officinarum L. Fifty-three polypeptides, representing about 3.6% of the total resolved spots, showed interspecific variation, whereas 78 polypeptides, about 5.3% of the total, showed intergeneric variation. Of the total polymorphic protein spots, qualitative (presence/absence) variation was more prevalent among the wild than among the cultivated species of the genusSaccharum, but the quantitative (spot intensity) variation was similar for both groups. The population of protein spots showing qualitative and quantitative variations was similar among the related genera of Andropogoneae. These polymorphic proteins can be used in genetic and evolutionary studies of the sugarcane genome.

Acidic ribosomal proteins of Dictyostelium discoideum that show electrophoretic similarity to Escherichia coli proteins L7 and L12.

This study documents the presence of three acidic proteins, A1 (pI 4.95), A2 (pI 4.85), and A3 (pI 4.70), in Dictyostelium discoideum ribosomes. All three proteins showed an apparent molecular mass of 13,000 by two-dimensional, sodium dodecyl sulfate gel electrophoresis. They were selectively released by treatment of ribosomes with 50% ethanol -1 M NH4Cl. The amino acid composition of A1, A2, and A3 were identical and indicated a predominant amount of alanine. All the above properties are shared by Escherichia coli proteins L7 and L12 and acidic ribosomal proteins in many eukaryotes. Unlike other eukaryotic systems, the acidic proteins of D. discoideum were found associated with the 40S rather than the 60S ribosomal subunit. Acidic proteins analogous in size and electrophoretic mobility to those of D. discoideum were also detected in several other cellular slime mold strains. Not one of the cellular slime mold acidic proteins reacted with antibodies to E. coli proteins L7 and L12 in immunodiffusion tests. In D. discoideum, the distribution of acidic proteins was altered during development. Amoebae contained all three proteins. In spores, A1 was absent and the relative amounts of A2 and A3 were lower than in amoebae. In addition, nine other acidic ribosomal proteins exhibited differences between vegetative amoebae and spores.

Unequal accumulation of 26S and 17S RNAs in ribosomes during spore germination in Dictyostelium discoideum.

Ribosome synthesis was studied in spores at the swelling stage and compared with freshly emerged and logarithmically growing vegetative amoebae. During the swelling stage of spore germination, ribosome synthesis was abnormal. Newly made ribosomes accumulated unequal amounts of 26S and 17S rRNAs. The stoichiometric ratio 26S:17S was 0.5 in swelling spores, compared with 0.9 in amoebae. The relative level of pre-rRNA persisting in the nucleus was apparently 2- to 3-fold higher in swelling spores than in amoebae. All of the known ribosomal proteins, except for a few, were made during the swelling stage and were associated with the newly made ribosomes in expected amounts. Analysis of the 2'-O-methyl ribose content in the newly made rRNAs suggest that methylation was defective in swelling spores. Compared with growing amoebae, the methyl content was 30 and 64% less in 26S and 17S RNAs from the swelling stage, respectively. It is suggested that undermethylation could be partly responsible for the differential accumulation of newly made 26S and 17S RNAs during the early stages of spore germination in Dictyostelium discoideum.

Salinity stress induced tissue-specific proteins in barley seedlings.

Protein changes induced by salinity stress were investigated in two barley cultivars, California Mariout, a salt-tolerant variety and Prato, a salt-sensitive variety. Rapidly growing young barley seedlings were exposed to NaCl and the newly synthesized proteins were resolved on two dimensional polyacrylamide gels following isoelectric focusing or nonequilibrium pH gradient gel electrophoresis in the first dimension. Salinity induces distinct protein changes in root and shoot tissues. In roots, the salinity effects are identical in both cultivars. First, salinity modulates the synthesis of two different sets of proteins, one of which is elevated, and the other, depressed. Second, six new proteins are induced all of which are low in molecular weight, 24 to 27 kilodaltons, with an isoelectric point range of 6.1 to 7.6. In contrast to roots, salinity induces cultivar-specific shoot proteins. Five new shoot proteins are induced whose molecular weights and isoelectric points fall within the range of 20 to 24 kilodaltons and 6.3 to 7.2, respectively. Three of the newly induced proteins are unique to Prato. In addition, salinity inhibits the synthesis of a majority of shoot proteins. The new proteins produced in roots and shoots are unique to each tissue and their induction is apparently regulated coordinately during salinity stress.

Differential mRNA transcription during salinity stress in barley.

The molecular and genetic bases of salinity tolerance in plants are not understood. Gene expression at the mRNA level was investigated in a salt-tolerant and a salt-sensitive genotype of barley. Seedlings were exposed to NaCl stress and translatable mRNAs were isolated from root and shoot tissues. A reticulocyte cell-free system was programed with barley mRNAs and the in vitro products were resolved on two-dimensional polyacrylamide gels following isoelectric focusing or nonequilibrium pH gradient gel electrophoresis in the first dimension. The functional mRNAs in unstressed seedlings were qualitatively almost indistinguishable in the two genotypes. However, salinity stress triggered differential transcription of specific mRNAs depending upon genotype and tissue. In roots, 12 new mRNAs were induced that encoded proteins of 21-34 kDa, with a pI range of 6.1-7.7. In shoots, the 9 new mRNAs coded for proteins of 18-50.5 kDa, with a pI range of 5.4-7.8. These new stress mRNAs represented one of two main classes. Class I consisted of mRNAs shared by both genotypes. Class II represented mRNAs specific to each genotype; unique mRNAs of roots accumulated preferentially in the salt-tolerant genotype, whereas those of shoots accumulated in the salt-sensitive genotype. The findings suggest that transcriptional as well as posttranscriptional mechanisms regulate gene expression in barley during salinity stress.

Protein synthesis in a maize callus exposed to NaCl and mannitol.

A maize (Zea mays, L) callus was exposed to media containing different levels of NaCl (0 to 3%) and mannitol (0 to 18.2%) for a period of 4 weeks, and the changes in growth and protein synthesis were determined. Cells are able to tolerate and grow in NaCl up to 1% (0.17 M) or mannitol up to 9.1% (0.5 M), but the relative overall growth rates are about 1/6 and 1/8 of the control, respectively. Protein synthesis, as assessed by pulselabeling of the cells with (35)S-methionine after exposure to the stress reagents at various times of incubation, suggests that the relative rates of amino acid uptake and its incorporation into proteins are inhibited as early as 4 h after exposure, and the extent of inhibition does not increase appreciably until after 1 week. Severe inhibition of uptake and protein synthesis results from prolonged exposures at growth-inhibitory concentrations of NaCl and mannitol. In general, the overall mean inhibition of cellular uptake and protein synthesis in the first 2-week period are approximately 50% and 35% for the NaCl (1%) and mannitol (7.3 %) treatments, respectively. No detectable differences are apparent in the abundant, steady state protein population as revealed by SDS-PAGE and on staining with Coomassie blue or silver, but random losses of individual proteins occur after 2 weeks at 2% and 3% NaCl and at 18.2% mannitol. Of the newly-synthesized proteins, discernible changes are found in 7 and 4 polypeptides in NaCl and mannitol treatments, respectively. Apparently three new proteins (74 kd, 28.5 kd, and 26.2 kd) are induced de novo under both treatments. Other proteins (39.5 kd, 39.0 kd, 30 kd, and 16.5 kd) show an increased or decreased level of synthesis. NaCl levels above 0.5% or mannitol levels above 3.6% do not after the pattern of newly-synthesized proteins. This altered expression of newly-made proteins in the maize callus occurs only after a week of exposure to salinity or osmotic stress and coincides with the cell growth phase.

Conservation and variation of ribosomal proteins in several species of the cellular slime molds Dictyostelium and Polysphondylium.

Genus- and species-specific composition of ribosomal proteins was investigated in four species of the genus Dictyostelium (D. discoideum, D. purpureum, D. murcoroides and D. giganteum) and two species of the genus Polysphondylium (P. pallidum and P. violaceum). Ribosomal proteins were resolved by a high-resolution, two-dimensional gel method. In general, the numbers and distributions for the majority of ribosomal proteins were similar within the species of each genus, although some differences were detected. More differences were observed between Dictyostelium and Polysphondylium than among the individual species within each genus. Stage-specific ribosomal proteins previously demonstrated in D. discoideum were found to be developmentally regulated in other Dictyostelium species, and in both Polysphondylium species. The study shows that ribosomal proteins may be a potentially useful new biochemical parameter for the molecular taxonomy of the cellular slime molds.

Enzymes of nucleic acid metabolism as biochemical markers of T cell development in mouse.

The levels of three purine salvage enzymes, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and 5'-nucleotidase (5'-N), which are known to be associated with certain immunodeficiency disorders were determined in mouse T lymphocytes. These enzymes showed characteristic changes depending on the stage of T cell development. The activity of ADA was 5-fold higher in thymocytes compared to splenic T cells. On the other hand, the splenic T cells displayed a 2-fold and a 4-8 fold greater activity of PNP and 5'-N, respectively than those of thymocytes. The apparent Km and Vmax values have been determined for all the 3 enzymes in the immature and mature T cells. The data demonstrate that the absolute and relative activity of these enzymes may be used as biochemical markers to characterize the T lymphocytes during different stages of differentiation.

Metabolic changes in ribosomes of Escherichia coli during prolonged culture in different media.

The metabolism of ribosomes during the exponential growth and post-exponential phase of Escherichia coli cells was investigated. Incubation of E. coli cells in two rich media: L-broth and phosphate medium, up to stationary phase shows no drop in viability or any changes in ribosomes. However, the survival rate during prolonged culture of the post-stationary-phase cells has been found to be a function of the incubation medium. The decline in viability is only slight in phosphate medium but very rapid in L-broth. So long as the viability is maintained, the level of ribosomes and the relative abundance of rRNA and ribosomal proteins in ribosomes of the post-stationary cultures are remarkably stable and are similar to exponentially growing cells. On the other hand, post-stationary cultures undergoing a rapid drop in cell viability lose 95% of the original ribosomes. These cultures accumulate a large pool of 30S and 50S subunits and a few 70S monosomes, all of which show deficiency in the various ribosomal proteins. No differences in rRNA can be detected but the number and the relative stoichiometry of individual ribosomal proteins are drastically altered. Only 13 of the 53 proteins known in the E. coli ribosome appeared in the same relative amounts as in the ribosomes of the exponentially growing cells. Six proteins (S12, S21, L2, L16, L20, L34) are completely lost and all others undergo partial loss. An analysis of the number and relative abundance of ribosomal proteins in the whole cells, as oppossed to isolated ribosomes, suggests that during the initial stages of the catabolism of ribosomes a crucial step is the formation of ribosomal-subunit-membrane complexes. The data emphasize the role of the constituents of ribosomes not only for the growth but also for the survival of E. coli cells. A model for the metabolism of ribosomes during the exponential growth and post-exponential phase of E. coli is presented.

Regulation of glycoprotein synthesis by thymic hormones in human thymocytes.

The synthesis of glycoproteins was studied in human thymocytes by metabolic labeling of cells with [3H] fucose and the influence of two thymic hormones, thymopoietin and thymosin fraction V was examined. Membrane and cytoplasmic proteins were prepared by a differential centrifugation procedure and resolved into distinct bands by sodium dodecylsulfate-polyacrylamide gel electrophoresis. De novo synthesis of about 10 membrane proteins and 6 cytoplasmic proteins was observed. Four of the membrane proteins, M1 (98Kd), M2 (45Kd), M3 (42Kd) and M5 (25.5Kd), appear to be similar to the cell surface glycoproteins identified in human thymocytes by others [Eur. J. Immunol. 10: 359 (1980)]. M2 (45Kd protein) was synthesized in thymocytes treated with thymopoietin, in cells from a patient with myasthenia gravis, and in a human T cell line. It was not made in normal thymocytes or thymocytes treated with thymosin. Hormones had no effect on the synthesis of cytoplasmic glycoproteins.

Unintegrated viral sequences in rat cells transformed by simian virus 40 and a temperature sensitive A gene mutant.

The status of viral sequences in rat cells transformed by simian virus 40 (SV40) and its temperature sensitive A gene mutant was investigated. Agarose gel electrophoresis of cell DNA prepared from clones picked from soft-agar and blot hybridization showed that sequences specific to SV40 genome were present both as integrated and unintegrated structures in rat clones. Digestion of rat cell DNA with various endonucleases with or without recognition sites in SV40 DNA and analysis indicated that the unintegrated viral genomes existed as full-length, covalently closed circular molecules. No differences in the free viral genomes were apparent between the clones transformed by the wild type and the mutant virus. The importance of the existence of free viral genomes in nonpermissive cells is discussed.

Activities of purine nucleotide metabolizing enzymes in human thymocytes and peripheral blood T lymphocytes: in vitro effect of thymic hormones.

Adenosine deaminase (ADA, E.C. 3.5.4.4) and purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1) activities are essential for the normal development and function of T lymphocytes. A comparison of the specific activity of these two enzymes and of ecto-5'-nucleotidase (5'-N, E.C. 3.1.3.5) of human thymocytes with that of peripheral T lymphocytes of children and young adults shows that significant differences exist between the activities of ADA and 5'-N in thymocytes and peripheral T cells. ADA activity is six-fold higher in thymocytes than in peripheral T cells whereas 5'-N activity is approximately one-fourth lower in thymocytes than in T lymphocytes. PNP activity is two-fold higher in T cells. The apparent Km and Vmax values for the three enzymes in thymocytes and peripheral T lymphocytes have been determined. The observed differences in enzyme activity are probably not entirely due to differences in the affinity of the enzymes for their substrates. The enzyme activities of a thymoma removed from a patient with myasthenia gravis had intermediate enzyme levels for ADA and PNP, but 5'-N was similar to peripheral T cells. Exposure of thymocytes in short-term culture to the thymic hormones thymopoietin (the pentapeptide TP5), thymosin fraction V or serum thymic factor (FTS) had no significant stimulatory or inhibitory effect on the activities of the three enzymes under our experimental conditions.

Ribosomal protein synthesis during spore germination and vegetative growth in Dictyostelium discoideum.

We have investigated the regulation of the synthesis of each ribosomal protein during spore germination and in vegetatively growing cells of Dictyostelium discoideum. Germinating spores and exponentially growing amoebae were labeled with [35S]methionine and the individual ribosomal proteins were resolved by electrophoresis on two-dimensional polyacrylamide gels. Functional mRNA levels for ribosomal proteins were analyzed by translating the slime mold RNA in a wheat germ cell-free system and separating the translation products by two-dimensional polyacrylamide gel electrophoresis. All slime mold ribosomal proteins were synthesized at every stage of spore germination and during vegetative growth; and the amount of synthesis of these proteins was highest at 1 to 2 h of germination. A comparison between the synthesis of ribosomal proteins in germinating spores with those in vegetatively growing cells suggests that although the majority of them seem to be similarly regulated, the regulation of the synthesis of some of the proteins may be different in both conditions. Although dormant spores lacked functional mRNAs for 16 ribosomal proteins, these mRNAs were detected as early as 1 h of germination. The mRNAs corresponding to all ribosomal proteins were present throughout germination, and their levels were highest at 2 h of germination. These results are consistent with the conclusion that the synthesis of the majority of ribosomal proteins during spore germination is regulated transcriptionally. However, vegetative amoebae lack three proteins (A, E, and L in our nomenclature), yet contain mRNA for these proteins that can be translated in vitro. Therefore, the synthesis of these proteins is apparently regulated translationally.