Lack of heat-shock response in preovulatory mouse oocytes

The response to heat (hs response) of preovulatory mouse oocytes was compared with that of mouse granulosa cells and characterized in regard to in vitro resumption of meiosis, amino acid incorporation into total protein, and qualitative analysis of protein synthesized before and after the shock. Granulosa cells displayed a hs response typical of other mammalian systems. When incubated at 43 degrees C for 20-40 min, these cells maintained a normal level of amino acid incorporation into total protein, responded to stress by new synthesis of 33- and 68-kDa heat-shock proteins (hsps), and enhanced synthesis of 70-kDa heat-shock cognate protein (hsc70) and of 89- and 110-kDa hsps. In contrast to granulosa cells, preovulatory mouse oocytes were very sensitive to hyperthermia. Incubation at 43 degrees C for 20-40 min strongly inhibited oocyte resumption of meiosis and protein synthesis and did not induce a new or enhanced synthesis of hsps. Unstressed preovulatory mouse oocytes constitutively synthesized 70- and 89-kDa polypeptides resembling hsc70 and hsp89 of granulosa cells.     

Changes in protein and amino acid content during anther development in fertile and cytoplasmic male sterile Petunia

Summary Development of anthers in cytoplasmic male sterile (CMS) Petunia diverges from the normal sequence of events early in meiosis. Quantitative and qualitative changes in morphology, proteins and free amino acid contents correlate with this divergence. In anthers of the fertile line (5719), total protein content increases, and SDS-PAGE protein patterns change as the anthers mature. Enhanced levels of three polypeptides with molecular weights of 64,000, 63,000 and 45,000 daltons characterize premeiosis in fertile anthers. Protein levels and patterns from anthers of the CMS line (5707) show little alteration during anther development. Protein synthesis seems to be at least partially blocked in the CMS microspore. The 63,000 and 45,000 dalton proteins are not present, and the absence of any unique protein(s) in the CMS line argues against a virus as the causal agent of CMS in Petunia. Analysis of free amino acids from anthers of the fertile line shows levels of proline and pipecolic acid 2–3 and 10–20 fold higher, respectively, than in the CMS line. The amino acids incorporated into proteins show no such differences; analysis of protein hydrolysates shows similar levels of each amino acid in both fertile and CMS lines at every developmental stage examined.     

Protein synthesis during hormonally induced meiotic maturation and fertilization in starfish oocytes

Uptake of radioactive amino acids and their incorporation into protein were examined during 1-methyladenine-induced maturation and subsequent fertilization of oocytes of the starfish Patiria miniata. The initial response to the hormone was a nearly immediate decrease in permeability to amino acids, indicating that the site of action of the hormone is on the cell surface. Protein synthesis began to increase starting about 12 min after 1-methyladenine stimulation and prior to germinal vesicle breakdown. It continued to rise throughout the first meiotic division. This protein synthesis was not required for assembly or initial functioning of the meiotic apparatus, although it was necessary for the completion of meiosis. Fertilization had no effect on the rate of protein synthesis.Oocytes of P. miniata provide an example of hormonal stimulation of protein synthesis in an invertebrate system.     

An autoradiographic and cytophotometric study of oogenesis in a pulmonate snail,Planorbarius corneus

Summary The spatial and temporal patterns of macromolecular syntheses in oocytes and somatic auxiliary cells of the snail Planorbarius corneus have been investigated by autoradiography and cytophotometry. Oogenesis has been divided into three stages, comprising early meiosis up to diplotene (stage I), previtellogenetic growth phase (stage II), and vitellogenesis (stage III). No DNA synthesis was found in any oocyte stage. In stage-I oocytes, only nucleoli were found labelled with ³H-uridine. Oocyte nuclei of stage II and III actively synthesize RNA in nucleoli and chromosomes. The most intense incorporation of uridine in chromatin probably occurs during the previtellogenesis — vitellogenesis transition period during which cytological findings suggest well developed lampbrush chromosomes. RNA synthesis in amphinucleoli of stage-III oocytes is restricted to basophilic nucleolar parts, whereas acidophilic parts (protein bodies) neither synthesize nor store RNA. During vitellogenesis oocytes incorporate amino acids into yolk platelet proteins. Radioactive proteins are found in yolk platelet precursors 5 h after injection of the tracer and in yolk platelets 3 h thereafter. The labelling pattern suggests that oocytes synthesize certain hitherto unidentified yolk components. No evidence for the participation of follicle cells in synthesis and transport of vitellogenic proteins has been obtained from autoradiography. Cytological findings suggest an important role for these cells in oogenesis. They are highly active in RNA and protein synthesis. Cellular differentiation is accompanied by polyploidization of the nuclei which attain a highest DNA content of 256 c. Polyploidization probably occurs in incremental steps as indicated by complete endomitotic chromosomal cycles. Autoradiographs show that, during vitellogenesis, oocytes do not incorporate significant amounts of glucose, and only certain follicle cells were labelled with glucose, probably indicating the synthesis of glycogen.     

Acetate Utilization and Macromolecular Synthesis During Sporulation of Yeast

Acetate utilization and macromolecule synthesis during sporulation (meiosis) of Saccharomyces cerevisiae were studied. When diploid cells are transferred from glucose nutrient medium to acetate sporulation medium at early stationary phase, respiration of the exogenously supplied acetate proceeds without any apparent lag. At the completion of ascospore development, 62% of the acetate carbon consumed has been respired, 22% remains in the soluble pool, and 16% is incorporated into lipids, protein, nucleic acids, and other cell components. Measurements of the rate of protein synthesis during sporulation reveal two periods of maximal synthetic activity: an early phase coincidental with increases in deoxyribonucleic acid, ribonucleic acid, and protein cellular content and a later phase during ascospore formation. Experiments in which protein synthesis was inhibited at intervals during sporulation indicate that protein synthesis is required both for the initiation and completion of ascus development.     

Amino acid uptake and protein synthesis in preimplanatation mouse embryos

Amino acid uptake and cycloheximide inhibitable incorporation into protein are demonstrable in follicular ova, unfertilized eggs, and in mouse embryos ranging from the 1-cell to the late blastocyst stages. The rates of uptake and incorporation are low and relatively constant until the early blastocyst (day 3) stage of development when they increase 3- to 9-fold. The rate of uptake continues to increase during the fourth day (late blastocyst stage) of development, but, despite embryonic growth, incorporation into protein remains constant. By exposing embryos to leucine and lysine at different concentrations, it is possible to dissociate the processes of uptake and incorporation into protein from one another and to use the latter as a measure of protein synthesis. Taking the number of embryonic cells into account, it is postulated that the period between 8- to 16-cell stage (day 2) and the early blastocyst stage is the only one in which the synthesis of major amounts of protein based on new messenger RNA synthesis is occurring.Leucine and lysine uptake take place by a facilitated process, although lysine transport is not readily saturated. Inhibitors of energy metabolism do not significantly affect amino acid uptake, but they do inhibit protein synthesis. However, since the rate of transport is highly temperature sensitive (Q₁₀ ? 3) and leucine is accumulated against a concentration gradient, active amino acid transport appears to be present.     

Germination of wheat embryos and the transport of amino acids into a protein synthesis precursor pool

Wheat (Triticum aestivum L. var. Lew) embryonic axes take up externally supplied radioactive amino acid (from a solution greater than 2 millimolar) such that the specific radioactivity of the total internal amino acid rapidly reaches that of the external solution. Nevertheless, incorporation of radioactive amino acid into protein increases steadily as the concentration of external amino acid is increased, indicating that the amino acid that is precursor to protein synthesis is not in equilibrium with the total internal amino acid pool. When the external source of amino acid is removed, incorporation of radiolabeled amino acid into protein continues at a rate comparable to that of embryos maintained in the radioactive solution. In explanation of these data, it is suggested that there are two separate cytoplasmic pools of amino acids, one a protein synthesis precursor pool, and the second, an expandable pool into which exogenous radioactive amino acids are taken up. The protein synthesis pool is fed at a limited rate from the expandable pool and at a far greater rate from an endogenous source. As a consequence, the specific activity of the amino acid that is the precursor for protein synthesis is considerably below that of the total internal pool and is determined by the rate of movement into the protein synthesis pool from the expanded radioactive cytoplasmic pool.

The rate of movement of amino acids from the expandable pool into the protein synthesis pool increases approximately 5-fold during the initial 4.5 hours of embryo germination. When this change is considered in analyzing the relative rates of protein synthesis, there is probably no more than a 2-fold increase in protein synthetic capacity between embryos germinated for 1.5 and 4.5 hours. The leveling off of the change in transport capacity after 4.5 hours suggests that the earlier increase in the rate of this process may be a necessary step before the embryos can begin to accelerate their growth rate.

     

Regulation of early mRNA synthesis after bacteriophage T4 infection of Escherichia coli.

Regulation of T4-specific mRNA synthesis was studied during leucine starvation of a leucine-requiring stringent Escherichia coli B strain. This was done by imposing starvation prior to T4 infection and then letting RNA synthesis proceed for different time periods. Rifampin or streptolydigin was added to stop further RNA synthesis, and protein synthesis was restored by addition of leucine. Samples were withdrawn at different times, and the enzyme-forming capacities found that, during conditions which elicit the stringent response in uninfected bacteria, immediate early mRNA is not stringently regulated. This conclusion contradicts the earlier conclusion of others, obtained by measuring incorporation of radioactive uracil; this is explained by the observation of Edlin and Neuhard (1967), confirmed and extended by us to the T4-infected cell, that the incorporation of uracil into RNA of a stringent strain is virtually blocked by amino acid starvation, whereas that of adenine continues at 30 to 50% of the rate seen in the presence of the required amino acid.     

Inhibition of Ribonucleic Acid Synthesis by Nalidixic Acid in Escherichia coli

The effect of low concentrations of nalidixic acid on ribonucleic acid (RNA) synthesis in Escherichia coli was examined. It was observed that RNA synthesis in exponentially growing cells was not significantly affected, in harmony with previous studies. However, RNA synthesis was markedly depressed by nalidixic acid during starvation for an amino acid or during chloramphenicol treatment. This effect was not caused by increased killing or inhibition of nucleoside triphosphate synthesis by nalidixic acid. The pattern of radioactive uracil incorporation into transfer RNA or ribosomes was not changed by the drug. The sensitivity of RNA synthesis to nalidixic acid in the absence of protein production may be useful in probing the amino acid control of RNA synthesis.     

BIOCHEMICAL CHANGES IN YEAST DURING SPORULATION. I. FATE OF NUCLEIC ACIDS AND RELATED COMPOUNDS

Changes in nuclear figures and in activities of nucleic acid and protein syntheses were observed mainly on Saccharomyces cerevisiae G2-2 during sporogenesis. Patterns of DNA synthesis and of meiosis show that the sporogenic process in yeast was divided into an induction phase (I-phase), a DNA-synthesizing phase (S-phase) and a maturation phase (M-phase). Meiotic figures appeared most frequently at the end of the S-phase at approximately 12 hr in sporulation culture. In M-phase visible spores formed. The amount of protein increased in the initial 7 hr culture of 1-phase, then decreased in the S- and M-phases. But in sporulation culture of the asporogenic diploid strain 3c × a, protein did not decrease. RNA increased within 3 hr of the I-phase then stopped increasing. DNA synthesis occurred critically during S-phase, i.e. between 7 and 12 hr. and was somewhat resumed during the later part of M-phase. Oligodeoxyri-bonucleotide content decreased in the I- and M-phases and increased temporarily. Deoxyribosides decreased linearly during the sporogenic processes. Based on these results and results of experiments estimating the incorporation of ¹⁴C-uracil into nucleic acid and ¹⁴C-amino acid mixture into protein fractions, the roles of nucleic acid synthesis activities in meiosis and in sporulation are discussed.     

Glyoxysomal malate dehydrogenase of watermelon cotyledons: De novo synthesis on cytoplasmic ribosomes

The development of glyoxysomal malate dehydrogenase (gMDH, EC 1.1.1.37) during early germination of watermelon seedlings (Citrullus vulgaris Schrad.) was determined in the cotyledons by means of radial immunodiffusion. The active isoenzyme was found to be absent in dry seeds. By density labelling with deuterium oxide and incorporation of [¹⁴C] amino acids it was shown that the marked increase of gMDH activity in the cotyledons during the first 4 days of germination was due to de novo synthesis of the isoenzyme. The effects of protein synthesis inhibitors (cycloheximide and chloramphenicol) on the synthesis of gMDH indicated that the glyoxysomal isoenzyme was synthesized on cytoplasmic ribosomes. Possible mechanisms by which the glyoxysomal malate dehydrogenase isoenzyme reaches its final location in the cell are discussed.Abbreviations mMDH mitochondrial malate dehydrogenase - gMDH glyoxysomal malate dehydrogenase - D₂O deuterium oxide - EDTA ethylenediaminetetraacetic acid, disodium salt     

MEASUREMENTS OF RATES OF PROTEIN SYNTHESIS IN RAT BRAIN SLICES

The use of tracer concentrations of labelled amino acids to measure incorporation in incubated slices of brain results in wide fluctuations with time in the specific activity of the precursor. Using concentrations of about 1 mm of labelled amino acid facilitates the accurate measurement of rates of synthesis. These higher precursor levels in the medium decrease the fluctuations in free amino acid specific activity due to dilution by endogenous amino acid and the production of amino acid by protein degradation, and decrease the lag in incorporation due to transport phenomena. Concentrations of 1 mm amino acid in the medium did not inhibit protein synthesis; with valine, leucine, phenylalanine, lysine and histidine, incorporation rates were similar when measured at trace concentrations and at 1 mm medium levels. The source of amino acid for protein synthesis appears to be intracellular. No evidence could be found for the preferential use of extracellular medium amino acid. The rate of incorporation of amino acids in incubated slices of rat brain was 0.087 per cent of the protein amino acid/h.     

Protein and Nucleic Acid Metabolism in Fruits: I. Studies of Amino Acid Incorporation During the Climacteric Rise in Respiration of the Avocado

Incorporation of (14)C L-valine and (14)C L-leucine into protein of tissue slices of the avocado fruit was relatively high during the early stages of the climacteric rise, declined sharply thereafter, and was virtually absent at the peak. The incorporation of amino acids in the preclimacteric stage was markedly lower than during the early stage of the respiratory rise. By following incorporation in relation to uptake at several concentrations it was established that the results were not a reflection of endogenous dilution.Puromycin was effective as an inhibitor of incorporation but not of oxygen uptake. When respiration was at its maximum there was no protein synthesis. It was concluded, therefore, that the respiratory upsurge characteristic of the climacteric was not related directly to protein synthesis.     

Kinetic analysis of amino acid pools and protein synthesis in amphibian oocytes and embryos.

Rates of protein synthesis have been measured in Rana pipiens oocytes and embryos and in Xenopus oocytes from the incorporation kinetics of two different concentrations of amino acid. This method does not require an independent measurement of the amino acid pools, since the pool size can be calculated directly from incorporation data. The effects of the concentration and diffusion of injected amino acid on the calculated values for amino acid pool size and flow rate are discussed. When the endogenous amino acid pool is appreciably expanded by the injected amino acid, the total amino acid pool in the oocytes or embryos may be considered as the precursor pool for protein synthesis. Under these circumstances, compartmentation of amino acids does not affect the results, except when lysine is used as tracer. The rates of protein synthesis in ovarian oocytes of Rana pipiens and Xenopus laevis are 18 and 50–54 ng/hr, respectively. In Rana pipiens, the rate increases 70% during maturation and another 50% before the two-cell stage. Finally, the rate approximately doubles between the two-cell and blastula stages.     

Protein synthesis and content of free amino acids in rat organs in experimental peritonitis

Experiments performed on rats shown that in peritonitis the mass of the animal's organs and C14-amino acid incorporation into tissue proteins is reduced. Free amino acid content in tissues and serum is increased. Decreased incorporation of C14-amino acid into hepatic proteins antecedes the increase of free amino acid content in this organ. In the kidneys, spleen and skeletal muscles diminished synthesis of proteins and increased protein catabolism occur simultaneously. It was shown that during the initial 24 hours of peritonitis the amino acid exchange between organs and systemic circulation are disturbed, however, the diminished synthesis of tissue proteins is not the result of free amino acid deficiency.     

Relative changes in incorporation rates of leucine and methionine during starvation survival of two bacteria isolated from marine waters

Abstract Two copiotrophic Gram-negative bacteria isolated from marine waters, S14 and Vibrio sp. DW1, were examined for changes in the rate of protein synthesis in the initial phase of energy and nutrient deprivation. The incorporation of [³H] leucine into the trichloroacetic acid (TCA)-insoluble material was examined as a method for estimating rates of protein synthesis. The incorporation of methionine was measured and compared with the results of leucine incorporation. Protein synthesis was demonstrated throughout a period of 120 h of starvation. The incorporation rate was related to the time of starvation and decreased subsequent to an initial increase during the first few hours of dormancy. Control experiments with proteinase K and chloramphenicol demonstrated that the labelled amino acids were preferentially incorporated into proteins. It was also demonstrated that the uptake of amino acids was not a rate-limiting step. During the first hours of starvation the ratio of the protein to the dry weight of the S14 cells increased parallel to the increase in the amino acid incorporation rate. The increased activity of the protein-synthesising system during the first hours of nutrient and energy depletion indicates the presence of an active cellular response to the downshift conditions. Furthermore, these findings are consistent with the increased respiratory activity during the first hours of starvation, which has previously been observed for the bacteria examined in this study.     

H3-phenylalanine incorporation into protein in the pituitary, hypothalamus and cerebral cortex of male rats during postnatal development.

Incorporation of H3-phenylalanine and concentration of protein in the anterior pituitary gland (APG), hypothalamus and cerebral cortex of male rats have been studied during postnatal development from day 21 to 120. APG and hypothalamus showed significantly higher rate of amino acid uptake and protein synthesis during the prepubertal phase, decrease at puberty and an age dependent variation thereafter. Protein concentrations and amino acid incorporation in the APG and hypothalamus are possibly influenced by the higher circulating levels of androgens during sexual maturation in marked contrast to their lack of effect on the cerebral cortex.     

Cytokinin effect on protein synthesis in vivo in higher plants

The influence of naphthalene-1-acetic acid and kinetin on protein synthesis in vivo was investigated by measuring the incorporation of radioactive amino acids into polypeptides of synthesizing polysomes. The second subculture of sterile pith tissue of Nicotiana tabacum L. cv. Wisconsin 38 grown on a medium containing only a minimum of growth substances was further starved in a small volume of medium lacking auxin and cytokinin for 12 h. An incubation period of 4 h with [¹⁴C]amino acids followed. The last 30 min of those incubations were carried out in the presence of actinomycin D and the last 20 min were performed under different conditions: a) without any growth substances, b) with naphthalene-l-acetic acid, c) with kinetin. By measuring the differences of the specific radioactivities of the polysomes the following results were revealed: 1) Kinetin increases the protein synthesis by an average of 35%-2) Auxin has no effect on protein synthesis.Abbreviations Act. D actinomycin D - NAA naphthalene-1-acetic acid Part of a Diplomarbeit, University of Bonn 1976