Temperature-sensitive breakdown in vivo of polysomes with mutants of Chlamydomonas reinhardii

Summary Two temperature-sensitive mutants of Chlamydomonas reinhardii Dangeard which are defective in protein synthesis were examined. Both show breakdown of their polysomes at the restrictive temperature into monosomes which do not contain fragments of mRNA. Many of the ribosomes still contain nascent peptides able to react with puromycin. The polysome breakdown involves only cytoplasmic (80S) ribosomes and is prevented or reversed when ribosome translocation is inhibited with cyloheximide.     

Analysis of Messenger RNA Coding for S100 Protein in the Mammalian Brain

S100 protein is a brain-specific protein which is absent at birth and first appears in rabbit brain 2–3 days after birth. To determine how the synthesis of this brain-specific protein is regulated, mRNA was isolated from brain polysomes and assayed for S100 protein mRNA activity by in vitro translation in a heterologous cell-free system and immunoprecipitation of released polypeptides with rabbit anti-S I00 protein antiserum. 5100 protein mRNA was detected primarily in small polysomes containing five to eight ribosomes, and virtually no S 100 protein mRNA was present in polysomes containing more than eight ribosomes. S100 protein mRNA was not detected in brain polysomes at stages prior to the induction of synthesis of S100 protein, i.e., in fetal brain or in 1-day neonates. The amount of S100 protein mRNA in polysomes of the cerebral cortex and cerebellum was measured to see if it correlated with the level of S100 protein in the two regions of adult brain. The cerebellum, which contained three to four times the level of S100 protein in the cerebral cortex, contained four times more S100 protein mRNA.     

Protein synthesis in oocytes of Xenopus laevis is not regulated by the supply of messenger RNA

The control of protein synthesis in oocytes of Xenopus laevis has been investigated by injecting oocytes with mRNA and polysomes followed by labeling with ¹⁴C-amino acid mixtures. Contrary to previous reports in which injected oocytes were labeled with ³H-histidine, injected globin mRNA is found to decrease amino acid incorporation into endogenous proteins competitively at all concentrations tested. No increase in overall amino acid incorporation is detected when more mRNA is supplied. Similar results are obtained after labeling injected oocytes with leucine, methionine, proline or valine individually. An explanation is presented for the conflicting results obtained when histidine is used as a label.When reticulocyte polysomes are injected, rather than purified globin mRNA, incorporation of amino acids into endogenous proteins remains roughly constant and overall incorporation increases. Similarly, when encephalomyocarditis viral RNA is injected together with either globin mRNA or reticulocyte polysomes, the globin mRNA causes decreased amino acid incorporation into encephalomyocarditis proteins, but the polysomes do not do so. The results demonstrate that different types of mRNA compete for a strictly limited translational capacity which is saturated in the normal oocyte. The limiting component is present in polysomes and is not message-specific. The constraint on protein synthesis in the amphibian oocyte cannot be fully explained by masked mRNA.     

On the role of the Escherichia coli RNA polymerase sigma factor in T4 phage development

Summary The rpoD800 mutation causing the temperature sensitivity of Escherichia coli RNA polymerase sigma factor has been used to demonstrate that the bacterial sigma factor is necessary throughout T4 phage development. In T4-infected RpoD800 mutant cells RNA synthesis is equally depressed at restrictive temperature at early and late stages of infection. The results show the bacterial sigma factor to be necessary for the synthesis of all RNA types in infected cells.     

Polysomal and non-polysomal messenger RNA in neuroblastoma cells: Lack of correlation between polyadenylation or initiation efficiency and messenger RNA location

Neuroblastoma cytoplasm was fractionated on sucrose gradients into polysomes (>90 S) and non-polysomal particles (<90 S). Purified RNA from these fractions was translated using a wheat germ lysate and translation products were compared by two-dimensional gel electrophoresis. Non-polysomal messenger RNA directed the synthesis of a specific subset of polysomal mRNA translation products. Careful comparison of individual translation products demonstrated that specific mRNAs were not randomly distributed between polysomes and the non-polysomal fraction.Fractionation of both RNA populations into polyadenylated (poly(A)⁺) and non-adenylated (poly(A)^?) species indicated that specific, abundant non-polysomal mRNAs were not less adenylated than their polysomal counterparts. Furthermore, comparison of translation products from assays of subsaturating and supersaturating RNA concentrations demonstrated that no simple correlation could be made between the relative initiation efficiency of a specific mRNA and its distribution between polysomes and non-polysomal particles.     

HSP 70 gene expression in Trypanosoma cruzi is regulated at different levels

The level of HSP 70 mRNA is altered in Trypanosoma cruzi cells incubated at supra-optimal temperatures: the total amount of this RNA per cell is increased at 37°C, and slightly decreased at 40°C relative to its level at 29°C. However, its amount is greater in the polysomes at either temperature. The relative increase of this RNA is larger in the polysomes fraction than it is in the total RNA. In addition the level of HSP 70 protein in heat-shocked cells is greater than would be expected from the recruitment of HSP 70 mRNA in the polysomal fraction. Taken together the data are interpreted as indicating that at 37°C and 40°C the HSP 70 gene regulation in T. cruzi involves both the selective accumulation of the HSP 70 mRNA in the polysomes and its preferential translation. At 37°C, in addition, an increase in the total amount of this template is observed in the cells.     

Activation of ribosomal and messenger RNA synthesis in excised Jerusalem artichoke tuber slices

RNA synthesis was studied in Jerusalem artichoke (Helianthus tuberosus L.) tuber slices immediately following excision and during the early period of aging in water. Incorporation of [³H]adenosine into RNA was detected as early as 20 min after excision. Measurement of the specific activities of RNA (cpm/g) and of ATP showed that RNA synthesis proceeded at a constant rate for the first several hours of aging and then increased moderately. [³H]adenosine was incorporated into polysomes throughout the aging period examined. Sucrose gradient fractionation of EDTA-dissociated polysomes showed that during the first 2 h of aging most of this incorporation was not into ribosome subunits but into presumed mRNA. Autoradiographic analysis of [³H]adenosine labelled nuclei showed that this was caused, at least in part, by a delay in the onset of rRNA synthesis synthesized during this time chromatographed as poly(A)-RNA on oligo(dT)-cellulose, indicating that a large part of the mRNA was not polyadenylated.     

Transition of Xwnt-11 mRNA from inactive form to polyribosomes in frogs during early embryogenesis

The distribution of Xwnt-11 mRNA between polysomes and informosomes was studied in Xenopus laevis and Rana temporaria during early embryogenesis. The ratio between polysomes and informosomes suggests their involvement in translation of these mRNAs. In eggs and immediately after fertilization the Xwnt-11 mRNAs are mostly positioned in informosomes. During the cleavage stage, these mRNAs have also been recognized in polysomes. Just before the onset of zygote genome functioning (at the stage of mid blastula), Xwnt-11 mRNA rapidly appears in polysomes of Rana embryos. However, in Xenopus, Xwnt-11 mRNA appears in polysomes only at the end of gastrula. Before this stage, the Xwnt-11 mRNA in Xenopus can be found mostly in informosomes.     

The effect of temperature onshibire ts cell clones in the compound eye ofDrosophila melanogaster

Summary We have analysed the effect of temperature on both developing and adult eye cell clones homozygous forshi ^ST139, a temperature-sensitive mutant ofDrosophila melanogaster. The mutant gene, autonomous in its cellular expression, causes structural modifications of ommatidial cells when adult clones of cells are exposed to the restrictive temperature (29°C) for several days. However, the mutant phenotype reverses to normal within 4 days at the permissive temperature (20°C). The results of pulse, shift-up and shift-down experiments show that the temperaturesensitive period for developing compound eye cells is from the late second instar up to the early pupa. Cytodifferentiation of compound eye cells is blocked by restrictive temperature treatment during this period, whereas cell proliferation does not seem to be directly affected. These results are discussed with regard to the other known aspects of the phenotype observed in mutant individuals.     

Genetic recombination of homologous plasmids catalysed by cell-free extracts of topo-isomerase mutant strains of Saccharomyces cerevisiae

Cell-free extracts of the yeast Saccharomyces cerevisiae can be used to catalyse the recombination of bacterial plasmids in vitro. Recombination between homologous plasmids containing different mutations in the gene encoding tetracycline resistance is detectable by the appearance of tetracycline-resistance following transformation of the recombinant plasmid DNA into Escherichia coli DH5. This in vitro recombination system was used to determine the involvement of eukaryotic topo-isomerases in genetic recombination. Cell-free extracts prepared from a temperature-sensitive topo-isomerase II mutant (top2-1) of S. cerevisiae yielded tetracycline-resistant recombinants, when the recombination assays were performed at both a non-restrictive temperature (30°C) and the restrictive temperature (37°C). This result was obtained whether or not ATP was present in the recombination buffer. Extracts from a non-conditional topo-isomerase I mutant (top1-1) of S. cerevisiae yielded tetracycline-resistant recombinants, as did a temperature-sensitive double mutant (top2-1/top1-8) at the restrictive temperature. The results of this study indicate that neither topo-isomerase I nor topo-isomerase II was involved in the recombinational activity examined.     

Temperature-sensitive Chinese hamster fibroblast mutant with a defect in RNA metabolism

We describe a new temperature-sensitive mutant of Chinese hamster cell fibroblasts. After a shift to the nonpermissive temperature of 40.5 degrees C, the rates of DNA, RNA, and protein synthesis declined rapidly (to < or = 50% within 12 h) and the progression of unsynchronized cells through the cell cycle was affected. We believe that DNA synthesis came to a halt after a short time, because cells no longer entered the S phase. The decrease in protein synthesis at 40.5 degrees C was shown to be a consequence of a decrease in the number of polysomes, whereas free 80S ribosomes accumulated. We concluded that the components of the protein biosynthetic machinery were intact (ribosomes and soluble factors), but synthesis was limited by a shortage of mRNA. The decline in mRNA production had a significant effect on the synthesis of proteins (e.g., heat shock proteins) translated from short-lived messages. We observed that both polyadenylated and nonpolyadenylated RNA syntheses declined at 40.5 degrees C, whereas the synthesis of small RNAs (4 to 5S) was less reduced. The argument is made that the temperature-sensitive phenotype is the result of a defect affecting mRNA synthesis.     

Regulation of yeast mitochondrial DNA synthesis

Summary A single recessive nuclear gene mutation has been isolated from strain 123.1 C of Saccharomyces cerevisiae which is conditionally deficient in mitochondrial DNA metabolism and has been termed tpi. Growth of this mutant strain in media containing galactose at 36°C causes a reduction of mitochondrial DNA synthesis as analyzed by incorporation of radioactive adenine into the mitochondrial DNA. These cells continue to grow and divide producing petite cells which are neutral and have been found to lack mitochondrial DNA as measured by radioactive incorporation of ³H-adenine into the mitochondrial DNA in the presence of cycloheximide at the permissive temperature. The rate of mitochondrial DNA synthesis of the mutant strain grown at the restrictive temperature in dextrose or glycerol containing media was found to be greatly reduced following two hours of exposure to the restrictive temperature. In addition, the action of this mutant gene has been found to be independent of the respiratory capacity of the mutant strain.     

Identification of globin mRNA in 10s RNA of rabbit reticulocytes

Electrophoresis on 6% polyacrylamide gels splits 10s RNA of detergenttreated polysomes from rabbit reticulocytes into two major bands. After these two RNAs are isolated separately, the first 10s RNA¹ directs the synthesis of both α and β chains in the Krebs II ascites cell-free system. In contrast, the second 10s RNA is inactive in directing globin synthesis. This result is further documented by separation of the two 10s RNAs by oligo dT-cellulose chromatography and by isolation of globin mRNA after EDTA-treatment of reticulocyte polysomes. Therefore, globin mRNA containing both α- and β-chain synthetic capacity moves as a single RNA species on electrophoresis in polyacrylamide gels.     

Protein synthesis with membrane-bound polysomes and albumin messenger RNA from livers of mutant mice

Summary Investigation of deficiencies in serum protein synthesis resulting from deletion-mutations at the albino locus in mice was continued usingin vitro conditions. Previous work showed that although total protein synthesis was only slightly lower in livers from albinos, newly synthesized protein appearing in plasma was 22% of that in controls. It was thought that the disorganized endoplasmic reticulum and Golgi apparatus, characteristic for the liver (and kidney) of these mutants, might be responsible for the observed deficiencies. In the present study mebrane-bound polysomes isolated from the livers of newborn albinos were 55% (c^3H/c^3H strain) and 62% (c^14CoS/c^14CoS strain) as efficient as those from normal littermates in incorporating radioactive leucine into protein in a cell-free system. These differences could not be eliminated by the addition of excess liver mRNA, exogenous soluble factors or by the exchange of cell sap between albino and control polysomes. In an earlier study albino liver slices synthesized only 13% (or 17% per mg of total protein synthesized) as much albumin as controls. We have now found that the level of albumin poly(A)⁺-RNA isolated from albino livers and assayed with a reticulocyte lysate, was almost as high (85%) as in controls. It was concluded that the very low level of albumin synthesis in albino livers did not result from a deficiency of albumin mRNA. Whether the rate-limiting step in synthesis of albumin in mutant livers is at the level of translation or processing for secretion requires further investigation.     

Rapid depletion of mutant eukaryotic initiation factor 5A at restrictive temperature reveals connections to actin cytoskeleton and cell cycle progression

Eukaryotic initiation factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid derived from the modification of lysine by spermidine. Two genes, TIF51A and TIF51B, encode eIF5A in the yeast Saccharomyces cerevisiae. In an effort to understand the structure–function relationship of eIF5A, we have generated yeast mutants by introducing plasmid-borne tif51A into a double null strain where both TIF51A and TIF51B have been disrupted. One of the mutants, tsL102A strain (tif51A L102A tif51aΔ tif51bΔ) exhibits a strong temperature-sensitive growth phenotype. At the restrictive temperature, tsL102A strain also exhibits a cell shape change, a lack of volume change in response to temperature increase and becomes more sensitive to ethanol, a hallmark of defects in the PKC/WSC cell wall integrity pathway. In addition, a striking change in actin dynamics and a complete cell cycle arrest at G1 phase occur in tsL102A cells at restrictive temperature. The temperature-sensitivity of tsL102A strain is due to a rapid loss of mutant eIF5A with the half-life reduced from 6 h at permissive temperature to 20 min at restrictive temperature. Phenylmethyl sulfonylfluoride (PMSF), an irreversible inhibitor of serine protease, inhibited the degradation of mutant eIF5A and suppressed the temperature-sensitive growth arrest. Sorbitol, an osmotic stabilizer that complement defects in PKC/WSC pathways, stabilizes the mutant eIF5A and suppresses all the observed temperature-sensitive phenotypes.     

Monocistronic messenger RNA in yeast

We have determined the rate of polypeptide chain synthesis on different size polysomes in yeast. The completion time for the average polypeptide chain in vivo at 23 °C is two minutes by this technique and is in good agreement with values we have determined by other independent methods.These kinetic experiments indicate that the average size of a nascent polypeptide chain on a polysome is directly related to the size of the polysome. This demonstrates that in the simple eucaryotic organism, Saccharomyces cerevisiae, mRNA is monocistronic in the sense that each mRNA molecule codes for one protein molecule which is released intact from the ribosome upon completion. The pattern of amino acid incorporation into Escherichia coli polysomes is distinctly different. These findings have a number of interesting implications for the genetics of the lower eucaryotes and indicate that the cellular mechanisms of control and co-ordination in yeast may differ from those found in procaryotes and may be similar to cellular mechanisms of control for mammalian cells.     

Labelling kinetics of RNA containing poly(a) in liver subcellular fractions

Kinetics of incorporation of (3H) uridine into cytoplasmic RNA fractions of rat liver is investigated. The fractions include free and membrane bound polysomes, rough membranes sedimenting with mitochondria and free cytoplasmic RNA particles. (1) Poly(A) containing RNA, isolated by oligo-dT cellulose, amounts to 0.4% of the total RNA in the homogenate, 0.5% in bound polysomes, 3.4% in free polysomes and 16% in free cytoplasmic RNA particles. (2) The rate of (3H) uridine incorporation into RNA lacking poly(A) proceeds uniformly in all subcellular fractions except for free cytoplasmic RNA particles, which accumulate negligible amounts of radioactivity. (3) The initial labelling of RNA containing poly(A) is most active in free cytoplasmic RNA particles supporting their identity as mRNA en route to polysomes. The initial specific radioactivities decrease in the following order: homogenate, bound polysomes, rough membranes sedimenting with mitochondria, free polysomes. The data suggest that mRNA is supplied to free and membrane-bound polysomes via different routes. The kinetic analysis indicates that free cytoplasmic RNA particles may be a precursor of mRNA of free polysomes rather than that of bound polysomes. (4) The kinetic differences of free and membrane bound polysomes are also demonstrated by comparing the radioactivity of RNA containing poly(A) to the total radioactivity at various incorporation times. In bound polysomes this decreases from 31% at 1 h to 10% at 25 h, whereas in free polysomes the corresponding ratio increases from 10 to 13%. RNA containing poly(A) of free cytoplasmic RNA particles represents 64% of the total radioactivity throughout the experiment.