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.

[High molecular weight transcript of the homologous to the LINE sequence in polysome poly(A)--RNA in the rat brain]. / Vysokomolekuliarnyi transkript, gomologichnyi posledovatel'nosti semeistva LINE v polisomnoi poli(A)--RNK mozga krysy.

Being used in blot-hybridization and S1-analysis, a number of cDNA clones homologous to L1 made it possible to detect a long abundant transcript of 7 kb. It accumulates solely in polysomal non-polyadenylated RNA from rat brain cells.

Combined use of oligo(dt) and 28S cDNA probes for the quantitation of total mRNA in polyribosomes: application to the castration-induced atrophy of the rat prostate.

The castration-induced atrophy of the rat prostate was used as a model for the validation of a sensitive technique allowing the quantitation of total mRNA in polyribosomes. Electron micrographs of polyribosome samples showed a decrease in polyribosomes length 7 days after castration (GDX). Specificity of labeled oligo(dt) probe for poly(A) was demonstrated and the technique was successfully applied to demonstrate that GDX is associated with a decrease in poly(A) mRNA content of polyribosomes. Provided that normalization of the hybridization signal for mRNA is achieved with a rRNA cDNA probe, the assay therefore represents a suitable tool for further studies regarding the translational regulation of total and/or specific mRNAs.

Stress hormones given to healthy volunteers alter the concentration and configuration of ribosomes in skeletal muscle, reflecting changes in protein synthesis.

1. The influence of elevated concentrations of stress hormones on the concentration of ribosomes and the relative proportion of polyribosomes, reflecting protein synthesis in vivo, in human skeletal muscle was investigated. Healthy volunteers were given a 6 h infusion of adrenaline (n = 8), cortisol (n = 8), a triple-hormone combination of adrenaline, cortisol and glucagon (n = 8), or saline (n = 8). 2. The total ribosome concentration declined by 30.4 +/- 7.2% in the triple-hormone group (P less than 0.01), by 26.9 +/- 8.6% in the cortisol group (P less than 0.05) and by 24.8 +/- 11.2% in the adrenaline group (P less than 0.05). The proportion of polyribosomes to total ribosomes decreased by 8.5 +/- 2.2% in the triple-hormone group (P less than 0.05). 3. During hormone infusion the serum glucose levels were enhanced. The insulin concentrations in serum were elevated in the adrenaline group and the triple-hormone group, but not in the cortisol group. Serum insulin decreased in the control group. 4. The results indicate an effect of the combined stress hormone infusion on the total ribosome concentration as well as on the relative abundance of polyribosomes. The single hormones influenced the total ribosome concentration only. The results suggest a critical role for stress hormones in producing the decline in muscle protein synthesis seen after trauma.

Polyadenylation and U7 snRNP-mediated cleavage: alternative modes of RNA 3' processing in two avian histone H1 genes.

The six chicken histone H1 genes have 3'-processing sequences typical of replication-dependent histone genes, which are expressed as poly(A)- mRNAs. However, by Northern analysis of RNA from several adult chicken tissues, as well as from embryonal skeletal muscle in vivo and in vitro, we have observed histone H1 transcripts longer than those predicted on the basis of the published genomic sequences. These RNAs are polyadenylated transcripts of the genes H1.01 and H1.10, which encode the 'c fraction' H1 protein subtypes. Both transcripts contain an internal stem-loop and purine-rich box associated with the 3' processing of poly(A)- histone mRNAs. The 2-kb poly(A)+ H1.01 transcript is present at high steady-state levels in tissues with low rates of DNA synthesis, has a longer half-life than the poly(A)- mRNA from the same gene, and is polyribosomal in embryonal skeletal muscle. The 1-kb poly(A)+ H1.10 RNA is the major H1.10 transcript in adult skeletal muscle. The properties of these RNAs suggest that they may contribute to the relaxed replication dependence of c fraction subtype expression. The polyadenylation signals of both genes are unusual in their association with processed (nonhistone) pseudogene-like elements, an arrangement with possible implications for the mechanism of alternative 3'-end formation in these genes.

Translational control of germ cell-expressed mRNA imposed by alternative splicing: opioid peptide gene expression in rat testis.

The three genes encoding the opioid peptide precursors (prodynorphin, proenkephalin, and proopiomelanocortin) are expressed in the rat testis. The sizes of the three opioid mRNAs in the testis differ from the sizes of the corresponding mRNAs in other rat tissues in which these genes are expressed. The smaller testicular proopiomelanocortin mRNA has previously been demonstrated to arise from alternative transcriptional initiation. In the present study, we found that the smaller testicular prodynorphin mRNA, expressed in Sertoli cells, results from alternative mRNA processing. Exon 2, which makes up 5' untranslated sequence, is removed from the mature transcript. Polysome analysis of brain and testis RNA indicates that the alteration of the prodynorphin leader sequence in the testis-specific transcript does not affect the efficiency of translation of this mRNA. The larger testicular proenkephalin transcript, expressed in developing germ cells, also results from alternative mRNA processing. Alternative acceptor site usage in the splicing of intron A results in a germ cell-specific proenkephalin transcript with a 491-nucleotide 5' untranslated leader sequence preceding the preproenkephalin-coding sequence. Polysome analysis indicates that this germ cell-specific proenkephalin mRNA is not efficiently translated. Mechanisms by which alternative mRNA splicing may serve to confer translational regulation upon the testicular proenkephalin transcript are discussed.

Alterations in polyadenylic acid-containing messenger RNA synthesis of brain polysomes after seizures of seizure-susceptible E1 mice.

The effect of seizures on synthesis of the polyadenylic acid (poly(A]-containing messenger RNA (mRNA) isolated from brain polysomes in a genetically seizure-susceptible E1 mouse was studied in vivo. The seizure in the E1 mice was induced by tossed-up stimulation. Immediately after the seizure ceased, the labeled orotic acid was injected into the brain. The incorporation rates of labeled orotic acid into poly(A)-containing mRNA isolated from polysomes are represented as the specific radioactivity (SR) (dpm/mg RNA) and the relative specific radioactivity (RSR) (dpm/mg RNA/dpm/mumoles of acid soluble uridine-5'-monophosphate (UMP]. Both the rates were reduced to 70% in SR and 65% in RSR at 1 h after the seizures. This reduction was gradually recovered to the level of interictal E1 mice at 6 h. The seizure-induced alterations are not attributable to the difference in the uridine nucleotide pool because the SR of UMP was not significantly affected by the seizure. The peak of labeled poly(A)-containing mRNA by analysis of gel electrophoresis displaced towards a lower molecular weight at 1 h after the seizures. The RNA showed a higher ratio of AMP and UMP per GMP and CMP in nucleotide composition, implying that this RNA is identical with DNA. These results suggest that the temporary decrease found in cytoplasmic mRNA synthesis induced by the seizures of E1 mice appears to be a result of impaired transcriptional processes in heterogeneous nuclear RNA synthesis and that the smaller mRNA coding for protein associated with seizures is newly synthesized during the postictal period.

Characterization of the translational defect to fiber synthesis in monkey cells abortively infected with human adenovirus: role of ancillary leaders.

Synthesis of the fiber protein of human adenovirus serotype 2 (Ad2) is 100-fold lower in abortively infected monkey cells, compared with productive infections, despite only a 5- to 10-fold reduction in fiber mRNA levels. Previously Anderson and Klessig (Proc. Natl. Acad Sci. USA 81:4023-4027, 1984) demonstrated a direct correlation between the productive nature of the infection, efficient synthesis of the fiber protein in vivo, and the presence of the x or y ancillary leaders on 10 to 25% of fiber messages. To determine at what level in translation these leaders might be important, the relative rate of initiation and elongation of each class of fiber message was assessed. The presence of the y ancillary leader in productively infected cells increased the rate of initiation about twofold, although translational elongation was similar on all fiber messages. However, the rate of elongation of all fiber messages was threefold slower in abortively infected than in productively infected cells. This reduced elongation rate in abortive infections was specific for fiber. The similar distribution of fiber mRNAs on polysomes in both infections suggests that initiation must also be partially blocked in abortive infections. Since the majority of the fiber mRNA even in productive infections did not contain the ancillary leaders, the initiation and elongation defects in the abortive infection cannot be fully explained by the absence of these leaders. Therefore, other factors in the infected cell must be influencing the rate of translation.

Immunological and chemical characterization of hamster brain polyribosomes-cytomatrix complexes.

We have studied the cytoskeletal nature of a brain subcellular fraction previously shown to contain polyribosomes. We have identified the major proteins of this fraction by electrophoretic comparison to a standard cytoskeletal fraction and by immunodetection. These methods have shown the presence of actin, glial fibrillary acidic protein, and neurofilament triplet proteins. We have also studied the effect of various ions and nonionic detergents on the stability of this structure. It was stable in presence of Triton X-100 up to 2% but disrupted by 200 mM K+ acetate.

Prosomes discriminate between mRNA of adenovirus-infected and uninfected HeLa cells.

Prosomes are small cytoplasmic RNP complexes associated with repressed mRNA. In in vitro translation, they discriminate between the mRNA of adenovirus-infected HeLa cells and those of uninfected cells grown under normal conditions. Prosomes as well as their RNA constituents interact much more strongly with poly(A)+ mRNA of infected cells and inhibit their translation in vitro preferentially. A possible role of prosomes in the differential regulation of translation is discussed.

A developmentally regulated gene product from Dictyostelium discoideum shows high homology to human alpha-L-fucosidase.

A cDNA library of poly(A+)-RNA has been prepared from membrane-bound polysomes of Dictyostelium discoideum and screened for clones hybridizing to mRNA species that encode developmentally regulated proteins. The clone investigated in this paper recognizes a 1.8 kb transcript that accumulates strongly between the growth phase and aggregation stage. Stimulation of cells with pulses of cAMP enhances the accumulation. The amino acid sequence derived from a complete cDNA and from a genomic clone displays extensive sequence identity to human liver alpha-L-fucosidase. The D. discoideum DNA sequence encodes a 50.5 kDa polypeptide with a hydrophobic signal peptide at the N-terminus. Antibodies against a synthetic peptide corresponding to amino acids 262-275 of the deduced protein sequence recognize a developmentally regulated 50 kDa protein in D. discoideum that is recovered in the particulate fraction.

Feedback control of ornithine decarboxylase expression by polyamines. Analysis of ornithine decarboxylase mRNA distribution in polysome profiles and of translation of this mRNA in vitro.

Cell growth and differentiation require the presence of optimal concentrations of polyamines. Ornithine decarboxylase (ODC) catalyses the first and rate-controlling step in polyamine synthesis. In studies using cultures of Ehrlich ascites-tumour cells, we have shown that the expression of ODC is subject to feedback regulation by the polyamines. A decrease in the cellular polyamine concentration results in a compensatory increase in the synthesis of ODC, whereas an increase in polyamine concentration results in suppression of ODC synthesis. These changes in ODC synthesis were attributed to changes in the efficiency of ODC mRNA translation, because the steady-state amount of ODC mRNA remained constant. We now show that the number of ribosomes associated with ODC mRNA is low, and that the increase in ODC mRNA translation takes place without a shift in the distribution of ODC mRNA towards larger polysomes. This finding indicates that the polyamines regulate the efficiency of ODC mRNA translation by co-ordinately affecting the rates of initiation and elongation. By analysing ODC mRNA translation in vitro, using a rabbit reticulocyte lysate, polyadenylated RNA from a cell line with an amplified ODC gene, and a monospecific anti-ODC antibody, we also show that spermidine, but not putrescine, exerts a direct regulatory effect on ODC synthesis.

Recovery of active ribosomal complexes from cellulose nitrate membranes.

The ternary Ac-[3H]Phe-tRNA-poly(U)-ribosome complex (complex C) [D. L. Kalpaxis, D.A. Theocharis, and C. Coutsogeorgopoulos (1986) Eur. J. Biochem. 154, 267-271] was used in model experiments aiming at the purification of this complex via adsorption on cellulose nitrate membranes and then desorbing the complex back into solution. The desorption was carried out at pH 7.2 in the presence of the nonionic detergent Zwittergent (ZW). The activity status of complex C was assessed with the aid of the puromycin reaction which characterizes ribosomal peptidyltransferase as part of complex C. The optimal conditions for desorbing complex C were 5 degrees C and a buffered solution containing 0.1% ZW. The kinetic constants of peptidyltransferase in the adsorbed state were kcat = 2.0 min-1, Ks = 0.4 mM. In the desorbed state, in solution, kcat = 3.4 min-1 and Ks = 0.3 mM. The method promises to be suitable for the rapid purification of ribosomal complexes containing mRNA and aminoacyl-tRNA.

Translational efficiencies of polyomavirus late mRNA molecules that differ in the sequences of their 5' noncoding late leader exons.

Mouse NIH 3T6 cells were coinfected with two strains of polyomavirus that differ only in the sequences of their 5' noncoding late leader exons. Polysomes were isolated at late times after infection and probed with oligonucleotides specific for each strain. Results indicate that the sequence of the late leader does not play a role in the translational efficiency of late polyomavirus messages.

Transcripts from the co-transposed segment of variant surface glycoprotein genes are in Trypanosoma brucei polyribosomes.

In Trypanosoma brucei the 5' proximal flanking sequences of a variant surface glycoprotein (VSG) gene, the co-transposed segment, are transcribed in a variant antigenic type- and stage-specific fashion along with the VSG gene. The precursor transcripts are subsequently processed to yield smaller transcripts from the co-transposed segment as well as the VSG mRNA. These co-transposed segment transcripts are quite abundant, polyadenylated and contain the spliced leader sequence, all characteristics of trypanosome mRNAs. We have found that all of the co-transposed segment transcripts from two VSG genes are present in polyribosomes. The nucleotide sequence of much of the co-transposed segment of one of these VSG genes, however, has no open reading frames coding for proteins longer than 49 amino acids. These results suggest that co-transposed segment transcripts do not encode essential proteins even though they are present in polyribosomes and may be translated.

Localization of a renal kallikrein immunoreactive-like substance in rat ureter.

An antibody against rat kallikrein was produced in rabbits and its localization was studied in various organs of the rat to confirm its specificity. The distribution of immunoreactive kallikrein was studied in rat ureter by use of immunochemical techniques. Ureteral tissue was fixed in Zamboni's-glutaraldehyde fixative and immunostained with indirect immunofluorescence and the peroxidase-antiperoxidase (PAP) method for light and electron microscopy. Preabsorption of the primary polyclonal antiserum with purified rat urinary kallikrein and substitution with normal serum were used as controls. By light microscopy, kallikrein was localized in the lamina propria and in the adventitial connective tissue surrounding the entire ureter. Immunoelectron microscopy confirmed this immunolocalization. Immunoreactive kallikrein was concentrated in fibroblasts of connective tissue and was not present in collagen fibers. Immunoreactivity was associated with the Golgi complex, free polyribosomes, and rough endoplasmic reticulum. No immunostaining was observed in other subcellular components of fibroblasts.

The use of two-dimensional gel electrophoresis in the analysis of organ-specific maize proteins.

Two-dimensional gel electrophoresis with non-equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate-polyacrylamide gels in the second dimension has been used for the analysis of organ-specific proteins in maize. The method has been used to study the whole protein pattern of developing organs and adult leaves as well as protein patterns of in vitro translation. Examples of two-dimensional immunoblotting and in vitro translation of endosperm-specific proteins are also shown. Two-dimensional gel electrophoresis appears as an essential analytical step in the identification of organ-specific proteins and for the detection of the protein products related to organ-specific genes identified by other means.

Synthesis of two proteins in chloroplasts and mRNA distribution between thylakoids and stroma during the cell cycle of Chlamydomonas reinhardii.

Chloroplasts contain thylakoid-bound and free ribosomes and polysomes. Whether binding of polysomes plays an immediate role in the regulation of chloroplast protein synthesis is not yet clear. In the present work, variations of protein synthesis and of mRNA content were measured not in greening, but in fully differentiated chloroplasts during the cell cycle of synchronized cultures of Chlamydomonas reinhardii. At different times of the vegetative cell cycle, the RNA was extracted from free and thylakoid-bound chloroplast polysomes and the partition of mRNAs between stroma and thylakoids was measured for two proteins, i.e. the 32-kDa herbicide-binding membrane protein and the soluble large subunit of the ribulose-1,5-bisphosphate carboxylase. At the same time the rates of synthesis of these two proteins were also determined. At 2 h after the onset of light, the content of both mRNAs in chloroplasts had doubled and 75-90% of each of these mRNAs were found to be bound to the thylakoids. The rate of protein synthesis, however, increased 10-fold, but reached its maximum only after about 6 h in the light. The differences in the time courses, in the stimulation of the rate of protein synthesis, and in the mRNA-binding to thylakoids point to a translational regulation of protein synthesis. Furthermore, since a very high proportion of polysomes were bound to thylakoids, containing mRNA for both a membrane and a soluble protein, this light-induced binding of polysomes to thylakoids seems to be an essential, but not the only, prerequisite for protein synthesis in chloroplasts.

Direct measurement of tubulin and bulk message distributions on polysomes of growing, starved and deciliated Tetrahymena using RNA gel blots of sucrose gradients containing acrylamide.

A method was developed using sucrose gradients containing acrylamide which greatly simplifies the measurement of the polysomal distribution of messages. After centrifugation, the acrylamide was polymerized, forming a "polysome gel". RNA gel blots of polysome gels were used to determine the polysomal distributions of alpha-tubulin and total polyadenylated mRNA in growing, starved (nongrowing) and starved-deciliated Tetrahymena and the number of messages loaded onto polysomes was calculated. These measurements indicated that the translational efficiencies of alpha-tubulin mRNA and total polyadenylated mRNA are largely unaffected when the rates of tubulin and total protein synthesis vary dramatically. Thus, differential regulation of alpha-tubulin mRNA translation initiation does not contribute to the greater than 100-fold induction of tubulin synthesis observed during cilia regeneration and in growing cells. The major translation-level process regulating tubulin synthesis in Tetrahymena appears to be a change in message loading mediated by a non-specific message recruitment or unmasking factor.