Absence Of Protein Synthesis Inhibitors Research Articles

A novel method for quantitative measurements of gene expression in single living cells.

Gene expression is at the heart of virtually any biological process, and its deregulation is at the source of numerous pathological conditions. While impressive progress has been made in genome-wide measurements of mRNA and protein expression levels, it is still challenging to obtain highly quantitative measurements in single living cells. Here we describe a novel approach based on internal tagging of endogenous proteins with a reporter allowing luminescence and fluorescence time-lapse microscopy. Using luminescence microscopy, fluctuations of protein expression levels can be monitored in single living cells with high sensitivity and temporal resolution over extended time periods. The integrated protein decay reporter allows measuring protein degradation rates in the absence of protein synthesis inhibitors, and in combination with absolute protein levels allows determining absolute amounts of proteins synthesized over the cell cycle. Finally, the internal tag can be excised by inducible expression of Cre recombinase, which enables to estimate endogenous mRNA half-lives. Our method thus opens new avenues in quantitative analysis of gene expression in single living cells.

Persistent long-term facilitation at an identified synapse becomes labile with activation of short-term heterosynaptic plasticity.

Short-term and long-term synaptic plasticity are cellular correlates of learning and memory of different durations. Little is known, however, how these two forms of plasticity interact at the same synaptic connection. We examined the reciprocal impact of short-term heterosynaptic or homosynaptic plasticity at sensorimotor synapses of Aplysia in cell culture when expressing persistent long-term facilitation (P-LTF) evoked by serotonin [5-hydroxytryptamine (5-HT)]. Short-term heterosynaptic plasticity induced by 5-HT (facilitation) or the neuropeptide FMRFa (depression) and short-term homosynaptic plasticity induced by tetanus [post-tetanic potentiation (PTP)] or low-frequency stimulation [homosynaptic depression (HSD)] of the sensory neuron were expressed in both control synapses and synapses expressing P-LTF in the absence or presence of protein synthesis inhibitors. All forms of short-term plasticity failed to significantly affect ongoing P-LTF in the absence of protein synthesis inhibitors. However, P-LTF reversed to control levels when either 5-HT or FMRFa was applied in the presence of rapamycin. In contrast, P-LTF was unaffected when either PTP or HSD was evoked in the presence of either rapamycin or anisomycin. These results indicate that synapses expressing persistent plasticity acquire a "new" baseline and functionally express short-term changes as naive synapses, but the new baseline becomes labile following selective activations-heterosynaptic stimuli that evoke opposite forms of plasticity-such that when presented in the presence of protein synthesis inhibitors produce a rapid reversal of the persistent plasticity. Activity-selective induction of a labile state at synapses expressing persistent plasticity may facilitate the development of therapies for reversing inappropriate memories.

CD40 ligand gene therapy directly induces apoptosis in carcinomas

14082 Background: CD40 is a member of the TNFReceptor superfamily expressed on a variety of immune cells. Interaction of CD40 with its ligand (CD40L) plays a key role in orchestrating immune responses. We have shown that CD40 is also expressed on a variety of carcinomas. CD40 ligation in this context can induce cancer cell death. Thus CD40 represents a rational target with direct cytotoxicity and induction of anti-tumour immunity. Methods: We generated an adenovirus encoding CD40L (AdCD40L) and investigated its effect in CD40-expressing carcinoma cells (bladder, ovary, hepatocellular) as follows:(i)comparison of Ad-delivered CD40L with recombinant soluble ligand (rsCD40L);(ii)inhibition of cleavage of CD40L from the cell membrane using metalloproteinase inhibitors (MMPI);(iii)generation of a mutant CD40L resistant to MMP cleavage. Results: (i)CD40 ligation in carcinomas stimulates survival and apoptotic signaling. rsCD40L is only cytotoxic in the presence of protein synthesis inhibition, which blocks survival pathways and allows apoptosis. In contrast, AdCD40L is cytotoxic even in the absence of protein synthesis inhibitor;(ii)AdCD40L results in expression at the cell membrane but this is cleaved from the membrane and detected as soluble ligand in supernatant of infected cells. Cleavage is inhibited by MMPI and this increases apoptosis, suggesting that membrane-bound CD40L is a more potent apoptotic stimulus than soluble ligand;(iii)we identified the cleavage site of CD40L and generated a mutant by deletion of this region and cloned this mCD40L into an adenovirus. AdmCD40L results in membrane expression of CD40L, with no detectable soluble CD40L, confirming its resistance to cleavage. AdmCD40L is significantly more cytotoxic than wild-type AdCD40L. Conclusions: The effect of CD40 ligation depends on the cellular context and the method of CD40L delivery: adenovirus delivery results in membrane-bound expression and this provides a stronger apoptotic stimulus than rsCD40L. Apoptosis is further enhanced by a mutant CD40L that is resistant to cleavage from the cell membrane. Thus AdmCD40L is a novel therapeutic that is a potent inducer of apoptosis. Studies are underway to assess these effects in vivo and to elucidate the contribution of immunostimulatory effects in syngeneic models. No significant financial relationships to disclose.

Potential role for a carbohydrate moiety in anti-Candida activity of human oral epithelial cells.

Candida albicans is both a commensal and a pathogen at the oral mucosa. Although an intricate network of host defense mechanisms are expected for protection against oropharyngeal candidiasis, anti-Candida host defense mechanisms at the oral mucosa are poorly understood. Our laboratory recently showed that primary epithelial cells from human oral mucosa, as well as an oral epithelial cell line, inhibit the growth of blastoconidia and/or hyphal phases of several Candida species in vitro with a requirement for cell contact and with no demonstrable role for soluble factors. In the present study, we show that oral epithelial cell-mediated anti-Candida activity is resistant to gamma-irradiation and is not mediated by phagocytosis, nitric oxide, hydrogen peroxide, and superoxide oxidative inhibitory pathways or by nonoxidative components such as soluble defensin and calprotectin peptides. In contrast, epithelial cell-mediated anti-Candida activity was sensitive to heat, paraformaldehyde fixation, and detergents, but these treatments were accompanied by a significant loss in epithelial cell viability. Treatments that removed existing membrane protein or lipid moieties in the presence or absence of protein synthesis inhibitors had no effect on epithelial cell inhibitory activity. In contrast, the epithelial cell-mediated anti-Candida activity was abrogated after treatment of the epithelial cells with periodic acid, suggesting a role for carbohydrates. Adherence of C. albicans to oral epithelial cells was unaffected, indicating that the carbohydrate moiety is exclusively associated with the growth inhibition activity. Subsequent studies that evaluated specific membrane carbohydrate moieties, however, showed no role for sulfated polysaccharides, sialic acid residues, or glucose- and mannose-containing carbohydrates. These results suggest that oral epithelial cell-mediated anti-Candida activity occurs exclusively with viable epithelial cells through contact with C. albicans by an as-yet-undefined carbohydrate moiety.

Induction of Murine Hepatocyte Death by Membrane-Bound CD95 (Fas/APO-1)-Ligand: Characterization of an In Vitro System

Hepatocytes constitutively express CD95 (also called Fas/APO-1) and are therefore potential targets for CD95-ligand (CD95L)-mediated injury. To study this mechanism of cell death in hepatocytes we developed an in vitro model of liver cell apoptosis using membrane-bound CD95L as the inducing agent. Primary mouse hepatocytes were cocultured with NIH 3T3 fibroblasts, stably transfected with mouse CD95L (FCD95L+). Fibroblasts stably transfected with vector only (FCD95L−) served as controls. Hepatocytes from mice expressing low levels of CD95 (Faslpr mice) served as controls for effects unrelated to CD95. Morphologic and biochemical studies indicate that CD95 is expressed in cultured mouse hepatocytes. Membrane-bound CD95 from transfected fibroblasts destroyed all cocultured hepatocytes within 24 hours in the absence of protein synthesis inhibitors. Characteristic features of apoptosis were observed in dying hepatocytes and occurred in the following sequence: formation of cytoplasmic blebs and nuclear condensation after 3 hours; nuclear fragmentation and DNA strand breaks after 4 hours. These changes were observed only when normal hepatocytes were cocultured with FCD95L+ and were not observed with FCD95L− or in hepatocytes from Faslpr mice. Anti-CD95 antibody (Jo2) evoked similar changes in hepatocytes, although to a much lesser extent. We conclude that coculture of mouse hepatocytes with FCD95L+ is a useful in vitro model for CD95-mediated apoptosis induced by CD95L. The high incidence of apoptosis caused by membrane-bound CD95L differs from the much smaller effects induced by the Jo2 antibody. In view of the high sensitivity of hepatocytes towards CD95L we speculate that CD95L-induced liver damage in vivo may be minimized by restricting exposure of hepatocytes to CD95L.(Hepatology 2000;32:779-785.)

The effect of temperature and protein synthesis on the renaturation of firefly luciferase in intact H9c2 cells.

A mild increase in temperature that does not exert an effect on tolerance development or synthesis of heat shock proteins (Hsps) in control cells can stimulate these processes when applied to cells that have previously been heat shocked. To study the underlying mechanism of this effect, H9c2 cells were stably transfected with the gene encoding firefly luciferase (Luc). Heat-shock-induced inactivation of Luc and its subsequent reactivation is frequently used as a model for cellular protein denaturation and renaturation. Luc reactivation was determined following a damaging heat shock (43 or 44 degrees C for 30 min) in cells that were subsequently exposed to either control temperatures (37 degrees C) or various mild hyperthermic conditions (from 38.5 to 41.5 degrees C for 1 h). To prevent changes in Luc activity consequent to new synthesis of Luc, Luc reactivation was monitored in the presence of cycloheximide, an inhibitor of protein synthesis. The results showed that reactivation of Luc was inhibited when heat-treated cells were post-treated under mild hyperthermic conditions. The observed increase in Hsp synthesis under mild hyperthermic post-heat shock conditions therefore appears to be the result of an increase in the period during which denatured proteins are present. In addition, we studied Luc reactivation in the absence of protein synthesis inhibitors. This condition led to much higher Luc activity. By estimating half-life times of Luc, the contribution of new Luc synthesis in this recovery could be determined, and only partially explained the observed increase in Luc reactivation after heat shock. Thus the synthesis of other proteins must be important for the renaturation of heat-damaged proteins.

The regulation mechanism of HLA class II gene expression at the level of mRNA stability.

The number of major histocompatibility complex (MHC) class II antigens may be regulated at different levels. Although transcriptional regulation has been studied most intensely, evidence for control mechanisms acting on the stability of MHC class II mRNAs has been reported. We have previously shown, in fact, that the half-life of MHC class II mRNA rapidly decreases in Raji cells upon inhibition of translation by cycloheximide; further data indicated that this effect was not correlated with the inhibition of the synthesis of trans-acting protein(s) required for mRNA stability. In the present work, we developed an in vitro mRNA decay assay system to measure HLA-DRA mRNA stability and used inhibitors of protein synthesis affecting different steps of the process of translation in order to discriminate among possible mechanisms determining controlled MHC class II mRNA hydrolysis. We found that HLA-DRA mRNA associated with polysomes derived from cells treated with either puromycin (which causes dispersion of polysomes and accumulation of monosomes) or cycloheximide (which slows down translation causing ribosome stalling) is more rapidly degraded than in the absence of protein synthesis inhibitors. On the basis of our findings, we suggest that arrest of protein synthesis per se exposes the HLA-DRA mRNA molecules to degradative activities co-sedimenting with the polysomal fraction.

Evidence for a Protein Synthesis-Dependent and -Independent TNFα Cytolytic Mechanism

The analysis of the effect of the protein synthesis inhibitors emetine (EM) or actinomycin D (ACT-D) on the TNFα-mediated cytolysis of L929 target cells demonstrates a bipbasic, concentration (10 -12-10 -4 M )-dependent curve indicative of two cytolytic mechanisms operative in L929 cells. One TNFα cytolytic mechanism is dependent on protein synthesis in the target cells, while the other cytolytic mechanism is protein synthesis independent. Both TNFα cytolytic mechanisms cause apoptosis (fragmentation of DNA) as shown by the TNFα-mediated release of tritiated thymidine, Apoptag, and DAPI staining, in the presence or absence of EM or ACT-D. The two cytolytic mechanisms are also similar in their requirement for lipoxygenase enzymes as shown by the ability of nordihydroguaiaretic acid (10 -6-10 -5 M) and ketoconazole (4 × 10 -6-2 × 10 -5 M) to block TNFα-mediated lysis of the target cells. However, the two cytolytic mechanisms differ in their requirement for the production of oxygen free radicals. The oxygen free radical scavengers, dimethylsulphoxide (0.2-0.4 M) and glutathione (2 × 10 -6-10 -5 M) block the TNFα-mediated cytolysis of target cells in the absence of protein synthesis inhibitors, but not in the presence of EM or ACT-D.

The Myxoma Virus TNF-Receptor Homologue (T2) Inhibits Tumor Necrosis Factor-α in a Species-Specific Fashion

The myxoma virus T2 protein shares extensive homology with the ligand binding domains of tumor necrosis factor (TNF) receptors. To characterize the T2-TNF interaction, myxoma T2 protein and rabbit, mouse, and human TNFα were expressed independently from vaccinia virus vectors. Growth of the TNFα-expressing viruses was significantly attenuated in TNF-hypersensitive L929-8 cells, and these cells were rapidly lysed by all three TNFαs. Rabbit cells showed strict species specificity in that RK-13 and SIRC cells were only sensitive to lysis by the homologous rabbit TNFα. Although RK-13 cells were hypersensitive to rabbit TNFα even in the absence of protein synthesis inhibitors, growth of T2 nonexpressing myxoma and vaccinia viruses in RK-13 cells was only modestly reduced in the presence of rabbit TNFα, suggesting that poxviruses possess additional anti-TNF mechanisms. When the ability of the myxoma T2 protein to inhibit biological activities of TNFα was assayed. T2 protein effectively protected L929-8 cells from lysis by rabbit, but not human or mouse TNFα. These studies show that rabbit TNF receptors may be species-specific for rabbit TNFα and we conclude that the myxoma T2 protein evolved to specifically inhibit TNF activities from its natural host, the South American rabbit.

D1 Protein Degradation and psbA Transcript Levels in Synechocystis PCC 6803 during Photoinhibition in vivo

The relation of photoinhibition of Photosystem II (PS II) to the rate of degradation of the reaction center protein D1 was studied in vivo in the cyanobacterium Synechocystis 6803. Exposure of cells to a PPFD of 1500 μmol m -2 s -1 induced 75% photoinhibition of oxygen evolution of PS II within 2 h. In spite of severe photoinhibition, only a slight net decrease was observed at the steady-state level of the D1 protein as determined by quantitative immunoblotting analysis. However, in the presence of protein synthesis inhibitors degradation of the D1 protein occurred rapidly and photoinhibition of PS II was accelerated. Pulse and chase experiments under strong illumination revealed that the D1 protein turned over rapidly with a half-life of about 30 min. The presence or absence of psbA mRNA carrying polyribosomes on the thylakoid membranes did not significantly affect the rate of D1 protein degradation. Our results indicate that no direct synchronization exists between degradation and synthesis of the D1 protein and it is probable that reassembly and activation of PS II after insertion of a new copy of the D1 protein to the PS II complex are the rate-limiting factors of the repair of photodamaged PS II in cyanobacteria. Northern blot analysis of psbA mRNA in the presence and absence of protein synthesis inhibitors demonstrated that protein factors are needed to regulate the expression of the psbA genes in Synechocystis 6803.

Comparison of the cytostatic and cytolytic activity of tumor necrosis factor-α and interleukin 1α in human malignant cell lines

The cytokines tumor necrosis factor-α (TNF-α) and interleukin 1α (IL-1α) share many properties, including in-vitro cytotoxicity. Because cytotoxicity can result from either cytolytic or cytostatic activity, and because differentiating between these activities may have clinical relevance, we determined the cytostatic and cytolytic activity of TNF-α and IL-1α for the human cell lines ME-180, SiHa (cervical carcinomas) and A375 (melanoma). Results of these analyses showed that IL-1α mediated cytostatic activity only for A375 cells. IL-1α was not cytolytic in the presence or absence of protein synthesis inhibitors. TNF-α was cytostatic for A375 and ME-180 cells, and although TNF-α was not cytolytic in the absence of protein synthesis inhibitors, it was cytolytic in the presence of protein synthesis inhibitors. These results suggest that the difference between the cytolytic and cytostatic activities of IL-1α and TNF-α may have therapeutic implications for the use of these biological response modifiers in the treatment of gynecological malignancies.

Induction of hyporesponsiveness to an early post-binding effect of tumor necrosis factor by tumor necrosis factor itself and interleukin 1.

Modulation of cellular responsiveness to tumor necrosis factor (TNF) was studied in the human SV-80 cells. A marked cytocidal effect is exhibited by these cells at about 4 to 8 h after application of TNF together with protein synthesis inhibitors. Sensitivity of the cells to TNF toxicity was shown to be markedly decreased following their pretreatment with TNF itself or with interleukin (IL) 1 in the absence of protein synthesis inhibitors. The SV-80 cells respond to TNF also with enhanced phosphorylation of the small heat-shock protein, HSP27. This TNF effect is much more rapid than the cytocidal effect; it is observed within minutes of TNF application. The response to this effect, just like the response to the cytocidal effect, is markedly decreased following preexposure of the cells to either TNF or IL 1. Responsiveness to both effects of TNF is regained at the same time, about 15 to 20 h following removal of TNF or IL 1. The decrease in responsiveness after pretreatment with TNF or IL 1 does not reflect an inability of the pretreated cells to bind TNF. Although there is an initial decrease in TNF binding after such pretreatment, it is fully reversed already about 5 h following removal of the cytokines. The rate of uptake of TNF by the pretreated cells is also normal. In view of the rapidity of the effect of TNF on the phosphorylation of HSP27, it seems likely that the observed hyporesponsiveness reflects impairment of an early step in a signaling pathway, perhaps common to both the stimulation of phosphorylation and the induction of cell death by TNF. By restricting the duration of the effects of TNF this desensitization mechanism may safeguard against harmful consequences of these effects.

Aryl hydrocarbon induction of rat cytochrome P-450d results from increased precursor RNA processing.

We have previously demonstrated that cytochrome P-450d mRNA accumulation is induced at a posttranscriptional level by 3-methylcholanthrene (MCA) in primary cultures of rat hepatocytes grown in serum-free hormonally defined medium. Using dactinomycin chase experiments in this culture system, we found that MCA had no effect on the P-450d mRNA half-life. In addition, induction of P-450d occurred both in the presence and in the absence of protein synthesis inhibitors. An analysis of nuclear precursors showed that the accumulation of the primary transcript of the P-450d gene was induced to the same extent as that of the mature mRNA after MCA treatment and that the pattern of accumulation of precursors differed between treated and control liver cells. Since P-450d induction is thought to be a receptor-mediated event, these data are consistent with a model in which a direct interaction occurs between the receptor-ligand complex and the primary transcript.

Aryl Hydrocarbon Induction of Rat Cytochrome P-450d Results from Increased Precursor RNA Processing

We have previously demonstrated that cytochrome P-450d mRNA accumulation is induced at a posttranscriptional level by 3-methylcholanthrene (MCA) in primary cultures of rat hepatocytes grown in serum-free hormonally defined medium. Using dactinomycin chase experiments in this culture system, we found that MCA had no effect on the P-450d mRNA half-life. In addition, induction of P-450d occurred both in the presence and in the absence of protein synthesis inhibitors. An analysis of nuclear precursors showed that the accumulation of the primary transcript of the P-450d gene was induced to the same extent as that of the mature mRNA after MCA treatment and that the pattern of accumulation of precursors differed between treated and control liver cells. Since P-450d induction is thought to be a receptor-mediated event, these data are consistent with a model in which a direct interaction occurs between the receptor-ligand complex and the primary transcript.

Autoregulation of tubulin expression is achieved through specific degradation of polysomal tubulin mRNAs

We have utilized protein synthesis inhibitors to investigate the autoregulatory mechanism that uses the concentration of unpolymerized tubulin subunits to specify tubulin mRNA content in animal cells. Puromycin and pactamycin, both of which remove RNAs from polysomes, completely unlink tubulin RNA content from the level of free subunits, whereas pretreatment of cells with cycloheximide, which traps mRNAs onto stalled polyribosomes, enhances the specific degradation of tubulin RNAs in response to increases in the subunit content. Moreover, in the absence of protein synthesis inhibitors, the tubulin RNAs that are lost from cells with elevated free tubulin subunit levels are those that are associated with polyribosomes. Further, β-tubulin mRNAs encoding a truncated translation product of only 26 amino acids (and that cannot be polyribosomal) are not substrates for autoregulation. We conclude that autoregulation of tubulin synthesis is achieved by specifically altering the stability of tubulin RNAs that are bound to polyribosomes.

The in-vivo occurrence of Z DNA.

The energetics of the B-Z transition of two different types of cloned alternating purine/pyrimidine DNA sequences have been analysed by a two dimensional electrophoretic technique. Since the transition between right handed and left handed forms of these polymers is detected by alterations of electrophoretic mobilities of topoisomers of the plasmid DNA molecules, the method is not dependent on Z-DNA binding ligands. The measurements reflect intrinsic properties of the DNA unperturbed by the free energy of binding such a ligand. Direct evidence from the analysis of topoisomer distributions is presented which shows that d(GC)n.d(GC)n sequence elements within an E. coli plasmid will adopt a Z conformation in-vivo under conditions of blocked protein synthesis. Evidence for the in-vivo occurrence of Z-DNA was not detected in plasmid DNA isolated from bacterial cells growing in the absence of protein synthesis inhibitors. A model is proposed for a function for the B-Z transition in ensuring the correct pairing of homologous chromosomes during meiosis.

Semliki forest virus mutants with temperature-sensitive transport defect of envelope proteins

Seven RNA-positive temperature-sensitive mutants of Semliki Forest virus were studied to find mutants with a defect in the transport of envelope glycoproteins to the host cell plasma membrane. Surface immunofluorescence of infected cells was recorded at the restrictive temperature (39°) and after shift to the permissive temperature (28°) carried out in the presence or absence of protein synthesis inhibitors. Preformed envelope proteins of two mutants, ts-1 and ts-7, were found at the plasma membrane only after shift to the permissive temperature. Staining of intracellular antigens after mild detergent treatment of ts-1 and ts-7 infected cells showed that at 39° the envelope proteins had reached th Golgi apparatus. Electron microscopy of ts-1 and ts-7 infected cells, maintained at 39°, showed that all the nucleocapsids were free in the cytoplasm. In ts-7, but not in ts-1, infected cells binding of nucleocapsids and virus budding started soon after the cultures had been shifted to the permissive temperature. First the budding was observed only at the Golgi and later also at the plasma membrane. Envelope proteins labeled at the restrictive temperature were efficiently incorporated into ts-7 but not into ts-1 virus particles, released after shift to 28°. We suggest that the viral glycoproteins have different “signals” for the transport and for the binding of nucleocapsid. In ts-7 both “signals” become functional after shift to 28°, whereas in ts-1 only the “transport signal” defect is reversible.

In Vivo Transcription and Protein Synthesis Capabilities of Bunyaviruses: Wild-Type Snowshoe Hare Virus and Its Temperature-Sensitive Group I, Group II, and Group I/II Mutants

The in vivo primary and secondary transcription capabilities of wild-type snowshoe hare (SSH) virus and certain of its temperature-sensitive (ts) mutants have been analyzed. The results obtained agree with in vitro studies (Bouloy et al., C.R. Acad. Sci. Paris 280:213-215, 1975; M. Bouloy and C. Hannoun, Virology 69:258-264, 1976; M. Ranki and R. Pettersson, J. Virol. 16:1420-1425, 1975) which have shown that bunyaviruses are negative-stranded RNA viruses with a virion RNA-directed RNA polymerase. The in vivo transcription studies have demonstrated that in the presence of protein synthesis inhibitors (puromycin or cycloheximide) SSH virus can synthesize viral complementary RNA (primary transcription) throughout the infection cycle. The increased levels of viral complementary RNA obtained in the absence of protein synthesis inhibitors (secondary transcription) were not markedly reduced if cells were pretreated with actinomycin D (5 mug/ml), alpha-amanitin (25 mug/ml), or rifampin (100 mug/ml), although progeny virus yields were reduced by up to 80% in the actinomycin D- and rifampin-treated cells. The in vivo transcription capabilities of SSH group I ts mutants at temperatures which were nonpermissive (40 degrees C) for virus replication gave values comparable to those obtained at permissive temperatures (33 degrees C). The SSH group I mutants appear, therefore, to be RNA-positive mutant types. When compared with their transcription capabilities at 33 degrees C, the in vivo transcription abilities of four SSH group II ts mutants (and one double group I/II ts mutant) were found to be more impaired at 40 degrees C than those of the SSH group I ts mutants or wild-type SSH virus at 40 degrees C, although the viral complementary RNA synthetic capabilities of these group II (and group I/II) mutants at 40 degrees C were significantly higher than their primary transcription capabilities (as measured at 33 degrees C in the presence of puromycin or cycloheximide). It was concluded, therefore, that these SSH group II (and double group I/II) ts mutants have an intermediate RNA phenotype. Hybridization studies using (32)P-labeled individual L, M, and S viral RNA species of SSH virus have demonstrated the presence of viral complementary RNA to all three species in extracts of cells infected with SSH ts II-30 and incubated at 33 degrees C (primary and secondary transcription) or 40 degrees C, a nonpermissive temperature for its replication. The results of pulse-labeled in vivo protein analyses indicated that greater quantities of intracellular N protein (coded for by S RNA [J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978]) than G1 and G2 polypeptides (coded for by M RNA [J. R. Gentsch and D. H. L. Bishop, J. Virol. 30:767-776, 1979]) were present in extracts of cells infected with wild-type SSH virus. In extracts of SSH group I, II, or I/II ts mutant-infected cells incubated at 33 degrees C, N and G1, and for the group II mutant-infected cells, G2, viral polypeptides were detected, whereas in extracts obtained from group I or II mutant virus-infected cells incubated at 40 degrees C, low levels of N and G1 polypeptides were evident.

The effect of beta-D-xylosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage in the absence of protein synthesis inhibitors.

Incorporation of [35S]]sulphate, [3H]glucose and [3H]serine into glycosaminoglycans and proteoglycans of embryonic-chicken sternum was measured in vitro in incubation medium containing 4-methylumbelliferyl beta-D-xyloside or p-nitrophenyl beta-D-xyloside at low concentrations, and in the absence of inhibitors of protein synthesis. Incorporation of sulphate was decreased by 80% in incubations in which 1mM-4-methylumbelliferyl beta-xyloside or 2.5 mM-p-nitrophenyl beta-xyloside was present; under these conditions, serum factors stimulated incorporation to only a small extent. When the concentration of the xyloside was decreased tenfold, incorporation of sulphate was inhibited by 60-70%, but when normal human serum or L-3,3',5-tri-iodothyronine or both were also added to the incubation medium, incorporation was markedly stimulated. Experiments in which [35S]sulphate and [3H]glucose were incorporated simultaneously, and enzymic analysis of glycosaminoglycans formed in such experiments, indicated that chondroitin sulphate formed in the presence of 0.1 mM-4-methylumbelliferyl beta-xyloside contained 30-40% less sulphate than did chondrotin sulphate synthesized in the absence of xylosides. Similar experiments, with [3H]serine instead of [3H]glucose, suggested also a 20-30% decrease in chain length of the chondroitin sulphate; this was confirmed by direct gel filtration of labelled glycosaminoglycans on a calibrated column. Incorporation of [3H]glucose or [3H]serine was stimulated by serum and tri-iodothyronine in parallel with incorporation of sulphate. The changes seen in the total chondroitin sulphate were mirrored in the major proteoglycan fraction, purified by isopycnic centrifugation of salt-extracted proteoglycans. The labelling pattern of chondroitin sulphate from this proteoglycan indicated that decreased sulphation of chondroitin sulphate was largely due to the inferior ability of short polysaccharide chains to accept sulphate, with some direct interference with transfer of sulphate to all chains. The results also suggested that the action of serum factors on synthesis of proteochondroitin sulphate is exercised at the level of either protein synthesis or transport to the sites of initiation of polysaccharide synthesis.

Biogenesis of chloroplast membranes in Chlamydomonas reinhardi: Chloroplast-controlled transfer of cytoplasmic proteins to the developing chloroplast membranes as visualized by quantitative radioautography.

The light-induced formation of the photosynthetic membranes (greening) in y-1 mutant of Chlamydomonas reinhardi requires synthesis of new proteins which become incorporated into the growing membranes. It has been shown previously (Eytan and Ohad, 1970) that proteins synthesized by both chloroplast and cytoplasmic ribosomes concur in the formation of functional photosynthetic membranes, indicating the presence of a mechanism permitting the specific transfer of membrane proteins synthesized in the cytoplasm into the chloroplast. Transfer of such proteins cannot yet be identified by the usual biochemical techniques unless they become part of the growing photosynthetic membranes. However, it is possible to follow their synthesis and translocation between the different cellular compartments by use of quantitative electron microscopic radioautography. In the present work, the radioautographic grain distribution among chloroplast, chloroplast membrane, nucleus, mitochondria, and the remainder of the cytoplasm (cytosol) was carried out following short radioactive pulse-labeling and chase during greening of dark-grown mutants in the presence or absence of protein synthesis inhibitors. The results indicate that transport of some of the proteins of cytoplasmic origin to their final location within the chloroplast is at least partially controlled by concomitant synthesis of proteins by the chloroplast ribosomes.