The programs of protein synthesis accompanying the establishment of alternative phenotypes in Candida albicans

Under the regime of pH-regulated dimorphism, stationary phase cells of the dimorphic yeast Candida albicans can be induced to form exclusively and synchronously ellipsoidal buds or elongate mycelia at the same temperature and in the same nutrient medium, the sole determinant of phenotype in this case being pH. Employing pH-regulated dimorphism, cells were pulse-labeled with [³⁵S]-methionine during three consecutive intervals encompassing the preevagination period, the period including evagination and phenotypic commitment, and the post-evagination period. Labeled polypeptides were analyzed by 2D-PAGE. Of the 374 polypeptides examined, the majority (237) did not differ significantly in relative incorporation between the three pulse periods and were similar between budding and mycelium-forming populations. Sixty polypeptides were labeled at negligible or relatively low levels during the first pulse period, but at significantly higher levels during the second and third or third pulse periods. All but one were similar between budding and mycelium-forming populations. Seventeen polypeptides were synthesized at relatively high levels during the first pulse period, but at reduced or negligible levels during the second and third or third pulse periods. All but one were similar between budding and mycelium-forming populations. Only two polypeptides were found to be associated exclusively with mycelium-forming cultures, two associated exclusively with budding cultures, and two enriched significantly in budding cultures of wild-type cells. Employing a variant, MD20, which forms buds at both low and high pH, it was demonstrated that only one mycelium-associated polypeptide and only one bud-associated polypeptide are phenotype rather than pH-specific. Limits to this method of phenotype comparison are outlined, and the unusual similarity rather than dissimilarity in the programs of gene expression between the diverging populations considered in terms of phenotypic regulation.     

The involvement of cell wall expansion in the two modes of mycelium formation of Candida albicans

When budding cells of Candida albicans are starved for 20 min and then diluted into fresh nutrient medium at 37 degrees C, pH 6.7, they form mycelia by two alternative modes. For cells with small buds, the bud expands apically, resulting in a transiently tapered daughter cell. With continued growth, the daughter cell tapers into an elongated mycelium. For cells with large buds, the bud completes expansion in the budding form, the mother cell and then the daughter bud evaginate, and the evaginations grow as mycelia. The present study investigates whether the temporal and spatial changes in the zones of wall expansion during bud growth are involved in the two modes of mycelium formation. Data are presented which demonstrate that the transition circumference which determines the two modes of mycelium formation and the transition circumference at which the active apical expansion zone shuts down are both 7 micron. This exact correlation suggests that starved cells with buds with a circumference of less than 7 micron form mycelia in the tapering mode due to the reactivation of the still present apical expansion zone, and that starved cells with buds with a circumference greater than 7 micron complete bud growth by general expansion due to the absence of the apical expansion zone at the time of starvation.     

A comparison of volume growth during bud and mycelium formation in Candida albicans: a single cell analysis

When stationary phase cells of the dimorphic yeast Candida albicans are diluted into fresh medium at pH 4.5 (low pH), they synchronously form ellipsoidal buds, but when diluted into the same medium at pH 6.7 (high pH), they synchronously form elongate mycelia. Using a perfusion chamber to monitor single cells, we have compared the rates of volume growth between budding and mycelium-forming cells. Results are presented which demonstrate that: (1) after release from stationary phase into medium of low or high pH, each original sphere grows in volume to the time of initial evagination, but does not grow subsequently; (2) successive budding on the original mother cell occurs without interruption resulting in continuous volume growth; however, an interruption in volume growth of the initial bud (B1) occurs before it in turn evaginates; and (3) the rate of volume growth of the first bud at low pH is identical to the rate of volume growth of the mycelium at high pH even though the surface to volume ratios are quite different. The last result is unexpected and is therefore considered in relation to cell wall deposition.     

The dependency of nuclear division on volume in the dimorphic yeast Candida albicans

When stationary phase cells of the dimorphic yeast Candida albicans are induced to synchronously form mycelia, over 90% of the cells undergo nuclear division. However, when stationary phase cells are induced to synchronously form buds, less than half undergo nuclear division even though all form buds. The majority of cells which do not undergo nuclear division form buds with volumes below a threshold value and the majority of cells which do undergo nuclear division form buds with volumes above this threshold value. In this report, we have investigated the possibilities that cells which form small buds do not attain a particular mass threshold. Cell cultures were examined for DNA replication, dry weight, and protein content during synchronous bud and during synchronous mycelium formation. Evidence is presented which indicates that the lack of nuclear division in over half of a budding population is due to low daughter cell volumes or to low surface areas, and not to their failure to attain a mass threshold or to replicate their DNA. The dependency of nuclear division on daughter cell volume is discussed.     

Growth and the inducibility of mycelium formation in Candida albicans: a single-cell analysis using a perfusion chamber

In Candida albicans, cells actively growing in the budding form cannot be immediately induced to form a mycelium until they enter stationary phase. However, if exponential phase cells are starved for a minimum of 10 to 20 min, they are inducible. Using a video-monitored perfusion chamber, we found that starved cells were able to form mycelia regardless of their position in the budding cycle. When starved exponential cells were released into fresh nutrient medium at high temperature and pH, conditions conducive to mycelium formation, unbudded cells evaginated after an average lag period of 75 min and then grew exclusively in the mycelial form. Depending upon the volume, or maturity, of the bud, budded cells entered two different avenues of outgrowth leading to mycelium formation. If the daughter bud was small, growth resumed by apical elongation of the bud, leading to a 'shmoo' shape which tapered into an apical mycelium. If the daughter bud was large, the cell underwent a sequence of evaginations: first, the mother cell evaginated after an average period of 75 min; then the daughter bud evaginated 40 min later. Both evaginations then grew in the mycelial form. In this latter sequence, the evagination on the mother cell was positioned non-randomly, occurring in the majority of cells adjacent to the bud. All buds undergoing evagination contained a nucleus, but roughly 20% of buds undergoing apical elongation did not.     

PsRBR1 encodes a pea retinoblastoma-related protein that is phosphorylated in axillary buds during dormancy-to-growth transition

In intact plants, cells in axillary buds are arrested at the G1 phase of the cell cycle during dormancy. In mammalian cells, the cell cycle is suppressed at the G1 phase by the activities of retinoblastoma tumor suppressor gene (RB) family proteins, depending on their phosphorylation state. Here, we report the isolation of a pea cDNA clone encoding an RB-related protein (PsRBR1, Accession No. AB012024) with a high degree of amino acid conservation in comparison with RB family proteins. PsRBR1 protein was detected as two polypeptides using an anti-PsRBR1 antibody in dormant axillary buds, whereas it was detected as three polypeptides, which were the same two polypeptides and another larger polypeptide 2 h after terminal decapitation. Both in vitro-synthesized PsPRB1 protein and lambda protein phosphatase-treated PsRBR1 protein corresponded to the smallest polypeptide detected by anti-PsRBR1 antibody, suggesting that the three polypeptides correspond to non-phosphorylated form of PsRBR1 protein, and lower- and higher-molecular mass forms of phosphorylated PsRBR1 protein. Furthermore, in vivo labeling with [³²P]-inorganic phosphate indicated that PsRBR1 protein was more phosphorylated before mRNA accumulation of cell cycle regulatory genes such as PCNA. Together these findings suggest that dormancy-to-growth transition in pea axillary buds is regulated by molecular mechanisms of cell cycle control similar to those in mammals, and that the PsRBR1 protein has an important role in suppressing the cell cycle during dormancy in axillary buds.     

Filament ring formation in the dimorphic yeast Candida albicans

Stationary phase cultures of Candida albicans inoculated into fresh medium at 37 degrees C synchronously from buds at pH 4.5 and mycelia at pH 6.5. During bud formation, a filament ring forms just under the plasma membrane at the mother cell-bud junction at roughly the time of evagination. A filament ring also forms in mycelium-forming cells, but it appears later than in a budding cell and it is positioned along the elongating mycelium, on the average 2 microns from the mother cell-mycelium junction. Sections of filament rings in early and late budding cells and in mycelia appear similar. Each contains approximately 11 to 12 filaments equidistant from one another and closely associated with the plasma membrane. In both budding and mycelium-forming cells, the filament ring disappears when the primary septum grows inward. The close temporal and spatial association of the filament ring and the subsequent chitin-containing septum suggests a role for the filament ring in septum formation. In addition, a close temporal correlation is demonstrated between filament ring formation and the time at which cells become committed to bud formation at pH 4.5 and mycelium formation at pH 6.5. The temporal and spatial differences in filament ring formation between the two growth forms also suggest a simple model for the positioning of the filament ring.     

Protein Synthesis in Isolated Plastids during Chloroplast Development in Wheat

Light-driven protein synthesis in isolated plastids was studiedduring the greening of etiolated wheat (Triticum aestivum L.)seedlings. The process was divided into five phases (I to V)according to the recovery of plastids from the leaf tissue.The activity was not detected in the etioplasts, but rapidlyincreased to the maximum level in phase I and remained at thislevel through phase II. During the transition from phase IIto III, the activity rapidly decreased to one-third and thencontinued to decrease slowly. The plastid polypeptides synthesizedduring the greening were analyzed by SDS-polyacrylamide gelelectrophoresis. In phase I, membrane polypeptides having molecularweights of about 21k were synthesized, while 23 k membrane polypeptidewas synthesized in phases III, IV and V. Synthesis of solublepolypeptides of 50–60 k and membrane polypeptides of 15k and 30–35 k was active in phases I and II, but decreasedbetween phases II and III. (Received October 31, 1983; Accepted May 14, 1984)     

Inhibition of the differentiation of Candida albicans by the chelator 1,10-phenanthroline

Several chelators were examined for their ability to prevent the synchronous release of 24- to 48-hour stationary phase singlet cells of the dimorphic yeast Candida albicans into either the mycelial or the budding phenotypes (in a defined liquid medium at 37°C; at pH 6.5 or pH 4.5, respectively). The only chelator that was found to inhibit mycelium formation completely and to restrict bud formation to about 10% was 1,10-phenanthroline at minimal concentrations of 50 M and 230 M, respectively.The inhibition of both phenotypes could be reversed completely by the addition of 200 M of ZnSO₄. The synchrony of recovery from inhibition by the addition of zinc paralleled that of the controls for both phenotypes, and the final number of mycelia or buds as a percentage of the control was the same (100%).These findings support the hypothesis that the lag period between the release from stationary phase and the onset of development for Candida represents the time of acquisition of a minimum threshold amount of a cation, such as zinc. The involvement of zinc in phenotypic development is discussed, suggesting that while zinc is involved in the initiation of development, it may not determine the phenotype of Candida albicans.     

Morphogenetic behavior of tropical marine yeast Yarrowia lipolytica in response to hydrophobic substrates

The morphogenetic behavior of a tropical marine Yarrowia lipolytica strain on hydrophobic substrates was studied. Media containing coconut oil or palm kernel oil (rich in lauric and myristic acids) prepared in distilled water or seawater at a neutral pH supported 95% of the cells to undergo a transition from the yeast form to the mycelium form. With potassium laurate, 51% of the cells were in the mycelium form, whereas with myristate, 32% were in the mycelium form. However, combinations of these two fatty acids in proportions that are present in coconut oil or palm kernel oil enhanced the mycelium formation to 65%. The culture also produced extracellular lipases during the morphogenetic change. The yeast cells were found to attach to the large droplets of the hydrophobic substrates during the transition, while the mycelia were associated with the aqueous phase. The alkane-grown yeast partitioned more efficiently in the hydrophobic phases when compared with the coconut oil-grown mycelia. A fatty acid analysis of the mycelial form revealed the presence of lauric acid in addition to the long-chain saturated and unsaturated fatty acids observed in the yeast form. The mycelia underwent a rapid transition to the yeast form with n-dodecane, a medium-chain aliphatic hydrocarbon. Thus, the fungus displayed a differential behavior towards the two types of saturated hydrophobic substrates.     

Differential profiles of soluble proteins during the initiation of morphogenesis in Candida albicans

Candida albicans is a dimorphic fungus that can grow either as yeast or as mycelia. The mycelial form may be required for tissue penetration and therefore may have a role in pathogenesis. The protein profiles of the cell-free S100 fraction from budding yeast cells and germ tube-forming cells (an early stage of the transition between yeast and mycelia) were evaluated using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Yeast growth or germ tube formation was induced in carbon-starved cells at 37° C by either glucose, galactose or N-acetylglucosamine at pH 4.5 or pH 6.7. More than 400 constitutively synthesised polypeptides were identified on 2-D PAGE by silver staining. A few polypeptides which seem to reflect the release from carbon starvation were detected, but no polypeptides unique to either morphology were observed. Fractionation of S100 preparations by polyethylenimine or heparin-agarose affinity chromatography, which have been used to detect DNA-binding proteins, revealed several proteins that were synthesised on the resumption of cell growth or in response to pH difference. Heparin-agarose also bound novel polypeptides in the size range 130–200 kDa that were preferentially synthesised in germ tube-forming cells. These results suggest that any protein factors that might exert a regulatory role early in germ tube formation are of low abundance, and that a minor group of soluble proteins involved in C. albicans morphogenesis may be differentially synthesised. Received: 11 March 1996 / Accepted: 10 July 1996     

A characterization of pH-regulated dimorphism in Candida albicans

When cells of the dimorphic yeast Candida albicans are grown to stationary phase in defined liquid medium at 25°C, they accumulate as singlets in G_l of the cell cycle. When these pluripotent, stationary phase singlets are released into fresh medium at 37°C, they synchronously evaginate after an average period of 135 to 140 minutes and form either buds or mycelia, depending upon the pH of the medium into which they are released. This method of dimorphic regulation offers the distinct advantage of comparability and serves as a very precise method for temporal comparisons of molecular and cytological events related to the establishment of the alternate growth phenotypes. In the present report, we have carefully examined the effects of individually varying pH or temperature on the length of the pre-evagination period, the population synchrony for evagination, and the phenotype of daughter cells. Exact phenotypic transition points, optima, and upper limits are defined for both temperature and pH. In addition, a method of pH-regulated dimorphism is developed in which the original temperature shift is removed from the inductive process. Finally, a common transition phenotype is described for cells reverting from the initial mycelial to budding phenotype when either pH or temperature traverse their respective transition points. The advantages as well as limitations of pH-regulated dimorphism are discussed in detail.     

Control of protein synthesis during blastocyst formation in the mouse.

In order to evaluate the dependence of the embryo on new mRNA synthesis during the period leading to blastulation, quantitative and qualitative aspects of protein synthesis in developing mouse morulae were investigated using α-amanitin, an inhibitor of RNA polymerase II. Only 1 of 423 early morulae cultured for 27 hr in the presence of 11 μg/ml α-amanitin cavitated, although most progressed as far as fully compacted morulae. About two-thirds of the untreated embryos cavitated during the same period. Incorporation of [³⁵S]methionine into protein was measured at 3- or 4-hr intervals over a 24-hr period and showed a two- to fivefold increase in control embryos. This increase was blocked in the α-amanitin-treated group although initial levels of incorporation were maintained. Total uptake of the amino acid appeared to be unaffected by the inhibitor. RNA synthesis, as measured by [³H]uridine incorporation over the same period, was reduced by between 5 and 52%, and the preblastulation surge in RNA synthesis was also blocked by α-amanitin. Two-dimensional polyacrylamide gel electrophoresis of labeled polypeptides synthesized by the embryos after 24-hr incubation in the presence or absence of the inhibitor revealed three distinct classes of polypeptide. The majority of polypeptides continued to be synthesized in the presence of α-amanitin whereas a small number of polypeptides, the synthesis of which would normally have increased during the development of the morula to the blastocyst, were prevented from doing so. A few polypeptides which normally cease to be synthesized over this period continued to be synthesized in the presence of α-amanitin. It is concluded that, while most of the proteins detectable at the morula stage are synthesized on mRNA templates of relatively long translational life, the general surge in protein synthesis, including the increased synthesis of a few species of polypeptide, are dependent on continuous translational activity.     

Functional Cell Types in Taste Buds Have Distinct Longevities

Taste buds are clusters of polarized sensory cells embedded in stratified oral epithelium. In adult mammals, taste buds turn over continuously and are replenished through the birth of new cells in the basal layer of the surrounding non-sensory epithelium. The half-life of cells in mammalian taste buds has been estimated as 8–12 days on average. Yet, earlier studies did not address whether the now well-defined functional taste bud cell types all exhibit the same lifetime. We employed a recently developed thymidine analog, 5-ethynil-2′-deoxyuridine (EdU) to re-evaluate the incorporation of newly born cells into circumvallate taste buds of adult mice. By combining EdU-labeling with immunostaining for selected markers, we tracked the differentiation and lifespan of the constituent cell types of taste buds. EdU was primarily incorporated into basal extragemmal cells, the principal source for replenishing taste bud cells. Undifferentiated EdU-labeled cells began migrating into circumvallate taste buds within 1 day of their birth. Type II (Receptor) taste cells began to differentiate from EdU-labeled precursors beginning 2 days after birth and then were eliminated with a half-life of 8 days. Type III (Presynaptic) taste cells began differentiating after a delay of 3 days after EdU-labeling, and they survived much longer, with a half-life of 22 days. We also scored taste bud cells that belong to neither Type II nor Type III, a heterogeneous group that includes mostly Type I cells, and also undifferentiated or immature cells. A non-linear decay fit described these cells as two sub-populations with half-lives of 8 and 24 days respectively. Our data suggest that many post-mitotic cells may remain quiescent within taste buds before differentiating into mature taste cells. A small number of slow-cycling cells may also exist within the perimeter of the taste bud. Based on their incidence, we hypothesize that these may be progenitors for Type III cells.     

Protein synthesis during the transition from the resting to the growing state in suspension cultures of Paul's Scarlet rose cells

Cells derived from Paul's Scarlet rose ( Rosa sp. ) were grown in the chemically defined medium of Nesius. When a stationary phase culture was diluted with fresh medium, growth was initiated after a pronounced lag period. DNA replication, as revealed by thymidine labeling and autoradiography, did not begin until 36 h, and mitotic figures were not observed until 48 h after dilution. A 10–15 fold increase in the rate of protein synthesis occurred during the lag period. This was brought about by a 3.5 fold increase in the amount of ribosomal RNA per cell, plus a doubling of both the percentage of ribosomes that are present as polyribosomes and the average number of ribosomes per polyribosome. The spectrum of polypeptides synthesized by these cells during the lag and early log periods of growth was examined. Polyribosomes were extracted from the cells at intervals preceding and accompanying the initiation of proliferative growth. The polyribosomes were translated in a wheat germ cell-free protein synthesizing system and the ³⁵S-methionine-labeled translation products were separated on polyacrylamide slab gels and by 2-dimensional gel electrophoresis. Comparatively few differences were observed between stationary phase, lag phase and log phase cells in terms of the spectrum of polypeptides synthesized in vitro. However, these various phases of the growth cycle could be characterized by a relatively high rate of synthesis of a few specific polypeptides. That is, while most proteins are synthesized throughout the growth cycle and even in non-growing cells at approximately the same relative rates, there are a few variable proteins whose synthesis marks a particular phase of the growth cycle.     

Changes in phenotypic expression in embryonic and adult cells treated with 5-azacytidine

We have previously shown that 5-azacytidine (5-Aza-CR) induced the formation of biochemically differentiated myotubes, adipocytes, and chondrocytes in the mouse embryo cell line, C3H/10T1/2CL8 (10T1/2), and that the induction of the muscle phenotype was cell cycle specific. Here we show that the adipocyte phenotype is also induced maximally in cells treated during early S phase. During this period, the minimum treatment time required for the subsequent formation of myotubes was 5 min and the number of myotubes formed was dependent on treatment time. The incorporation of ¹⁴C-5-Aza-CR into DNA during the cell cycle, however, was not enhanced during early S phase, suggesting that incorporation of 5-Aza-CR into specific DNA sequences synthesized during early S phase may be required for the expression of the new phenotypes. Single cells, obtained by plating cell suspensions into 16 mm wells at limiting dilution, were treated with 5-Aza-CR during S phase. The resulting clones showed a high frequency of phenotypic conversion, indicating that 5-Aza-CR did not act via a selective mechanism, and several of the clones were capable of expressing more than one phenotype. The cells required more than 2 division cycles after treatment with the analog for the expression of the muscle phenotype and the capacity to differentiate was retained for long periods of time in the absence of cell division. The adult mouse line, CVP3SC6, differentiated into functional striated muscle cells following treatment with 5-Aza-CR. The analog also caused oncogenic transformation in the adult line at the same concentration that was effective at inducing myogenic expression.     

Phase-Specific Polypeptides and Poly(A) RNAs during the Cell Cycle in Synchronous Cultures of Catharanthus roseus Cells

This study shows an overall analysis of gene expression during the cell cycle in synchronous suspension cultures of Catharanthus roseus cells. First, the cellular cytoplasmic proteins were fractionated by two-dimensional gel electrophoresis and visualized by staining with silver. Seventeen polypeptides showed qualitative or quantitative changes during the cell cycle. Second, the rates of synthesis of cytoplasmic proteins were also investigated by autoradiography by labeling cells with [³⁵S]methionine at each phase of the cell cycle. The rates of synthesis of 13 polypeptides were found to vary during the cell cycle. The silverstained electrophoretic pattern of proteins in the G₂ phase in particular showed characteristic changes in levels of polypeptides, while the rates of synthesis of polypeptides synthesized during the G₂ phase did not show such phase-specific changes. This result suggests that posttranslational processing of polypeptides occurs during or prior to the G₂ phase. In the G₁ and S phases and during cytokinesis, several other polypeptides were specifically synthesized. Finally, the variation of mRNAs was analyzed from the autoradiograms of in vitro translation products of poly(A)⁺ RNA isolated at each phase. Three poly(A)⁺ RNAs increased in amount from the G₁ to the S phase and one poly (A)⁺ RNA increased preferentially from the G₂ phase to cytokinesis.     

Biogenesis and light regulation of the major light harvesting chlorophyll-protein of diatoms

The apoprotein of the major light harvesting pigment-protein complex from the diatom Phaeodactylum tricornutum (UTEX 646) is composed of two similar polypeptides of 17.5 and 18.0 kilodaltons (kD). The in vivo synthesis of these polypeptides is inhibited by the 80s protein synthesis inhibitor cycloheximide, but not by the 70s ribosome inhibitor chloramphenicol. When total poly(A)⁺ RNA was used in in vitro protein synthesis, a number of polypeptides were synthesized with a dominant product at 22 kD. When the polypeptides were immunoprecipitated with monospecific antibodies to the 17.5 and 18.0 polypeptides, a single protein zone of 22 kD was detected. Immunoprecipitation with preimmune serum failed to precipitate detectable levels of protein at any relative molecular weight (M_r). These findings indicate that the two apoprotein polypeptides of the diatom light harvesting pigment-protein are translated from polyadenylated message on cytoplasmic ribosomes as either a single or two (or more) similar M_r precursor proteins. These findings also suggest that this protein is encoded in the nucleus.

Photosynthetic light adaptation features of P. tricornutum UTEX 646 indicate that it responds to low light by increasing cell size and numbers of photosystem I and II reaction centers per cell, but does not change photosynthetic rate per cell or photosynthetic unit sizes significantly. When low light cells are exposed to higher photon flux densities, the in vivo incorporation of label into the apoprotein of the light harvesting complex decreases. In contrast, high light grown cells show rapid (<3 hour) increases in apoprotein synthesis when exposed to low light levels. This is the first demonstration of a specific role of photon flux density in regulating the synthesis of a major light harvesting pigment-protein during photosynthetic light adaptation.

     

Synthesis of DNA-binding proteins during the cell cycle of WI-38 cells

Synthesis of DNA-binding proteins was investigated in WI-38 human diploid fibroblast cultures after stimulation with serum containing medium. Density-inhibited confluent monolayers of young (phase II) and aging (phase III) WI-38 cells can be stimulated to synthesize DNA by replacing the medium with fresh medium containing 10% fetal calf serum. Of the phase II cells, 35–50% showed a partially synchronized burst of DNA-synthesizing activity between 15 and 24 h whereas only 4–6% of phase III cells showed DNA-synthesizing activity at 20 h, and that cell fraction was increasing even at 38 h. This suggests either an extremely prolonged G 1 in stimulated phase III cells, or a heterogeneity of the population (e.g., a mixed population of pre- and postmitotic cells) for phase III cells. At various times after the change of medium, DNA-binding protein synthesis was examined in these stimulated cultures. Protein of mol. wt 20 000–25 000 D accumulated rapidly during early G 1 and declined thereafter, whereas larger protein (40 000 and 68 000 D) accumulated during the late G 1 or G 1-S transition period indicating that accumulation of these proteins is associated with the onset of DNA synthesis in the serum-stimulated cells. In cultures where the DNA synthesis has been reduced or inhibited by an excess of thymidine, hydroxyurea or dibutyryl cAMP, the accumulation of the larger proteins (40 000 and 68 000 D) was neglible as compared with non-stimulated cultures. Hydrocortisone did not exert any effect on the DNA-binding protein synthesis in phase II cells. However, it seems to increase the cell fraction which can respond to the serum factor in phase III cells as evidenced from the pattern of DNA-binding proteins synthesis.     

Stimulation of polypeptide synthesis by spermidine at the level of initiation in rabbit reticulocyte and wheat germ cell-free systems

It is shown that the stimulation of eukaryotic polypeptide synthesis by spermidine is due to the stimulation at the level of initiation by following reasons. The incorporation of formylmethionine into polypeptides was stimulated by spermidine at the same degree to the incorporation of leucine into polypeptides. Fluorography of the polypeptides formed showed that the number of chains of individual protein synthesized was larger when spermidine was added. The formation of the complex of Met-tRNA_f, globin mRNA and 40-S ribosomal subunits was stimulated by spermidine.