Glorin-regulated protein synthesis in Polyspondylium violaceum

Protein synthesis by Polyspondylium violaceum in response to the chemoattractant glorin was analyzed by two-dimensional gel electrophoresis to determine if glorin can affect gene expression. When cells developing in a shaking suspension culture were given glorin, five proteins exhibited significantly increased and three proteins exhibited decreased incorporation of L-[35S]methionine. Glorin was active from 10-1000 nM, a concentration range within which cells are chemotactically active. The extent of the response was dependent on the concentration and the length of exposure to glorin. This evidence suggests that glorin may act in part to mediate changes in gene expression during development.     

Interactions of Microbotryum violaceum (ustilago violacea) with its host plant Silene alba

Sex expression in Silene alba is determined genetically but can be changed in female plants upon infection with the heterobasidiomycete Microbotryum violaceum. This change is not caused by steroids, the classical plant growth regulators, nor by a diffusible morphogen produced and secreted by the fungus. Nor is the production of stamnes in genotypically female flowers caused by the transmission, incorporation and expression of a fungal plasmid since plants regenerated from diseased tissue of genotypically female S. alba did not yield stamen-producing flowers. Neither density gradient centrifugation nor agarose gel electrophoresis of endonuclease restricted DNA from M. violaceum revealed the presence of a plasmid. Southern blots of DNA from S. alba probed with labeled DNA of M. violaceum, however, indicated the presence of homologous, unique sequences absent in non-host plants. Since the same homologous sequences were identified in male and female S. alba, these DNA fragments are not homologous to the coding sequences for male sex expression in S. alba unless they represent genetic elements of the hypothetical gyndyioecious precursor. Two other aspects of the S. alba-M. violaceum interaction have yielded interesting results. M. violaceum grows as sporidia outside of the host, but as short hyphae in planta. The switch from sporidial to hyphal growth is mediated in vitro by hyphal growth factors (HGFs) isolated from aqueous host plant extracts as well as by -tocopherol. In addition to changing the fungal growth form. HGFs may serve as host recognition factors. Siderophore mutants of M. violaceum that accumulated less rhodotorulic acid than wild type also showed reduced or no pathogenicity, indicating that siderophores are an important factor in the host-pathogen interaction.     

Cyclic nucleotides and cyclic nucleotide phosphodiesterase during development of Polysphondylium violaceum

Polysphondylium violaceum is shown to produce and excrete cyclic nucleotides and to produce a cell-associated cyclic nucleotide phosphodiesterase(s). The amount of adenosine 3′,5′-cyclic monophosphate (cAMP) excreted by the amebae reaches a maximum during development when aggregation centers are just forming and then falls off rapidly. Measurements of total cAMP show that the amount synthesized increases more than 15-fold throughout development with the majority of the increase coming during the culmination stages. Guanosine 3′,5′-cyclic monophosphate (cGMP) is either not excreted or is excreted at levels below our limits of detection. An increase in the total cGMP synthesized occurs at mid-aggregation when two or three sharp peaks of synthesis are observed. However, development of P. violaceum is not affected by the addition of high concentrations of either cAMP or cGMP (or their dibutyryl derivatives) to the medium despite the fact that the cells produce these nucleotides. Cell-associated cyclic nucleotide phosphodiesterase activity, which hydrolyses both cAMP and cGMP, is greatest at the onset of starvation with a second increase in activity during aggregation.     

Control of membrane protein synthesis in bacillus subtilis

In synchronous cultures of Bacillus subtilis 168/S grown on succinate as a sole carbon source (mean generation time 115 min), chromosome initiation occurs at the beginning of the cell cycle but the rate of membrane protein synthesis doubles in mid-cycle more or less coincident with nuclear segregation. In glucose-grown cultures, the doubling in rate of membrane protein synthesis occurs at about the same time as nuclear segregation and DNA initiation at the beginning of the cycle. Control of the rate of membrane synthesis by the chromosome has been demonstrated by inhibiting DNA synthesis using thymine starvation and showing that membrane protein synthesis continues at a constant rate, whereas the rate of cytoplasmic protein synthesis almost doubles.I suggest that the replication of a region at or close to the chromosome terminus is required to allow the doubling in rate of membrane synthesis.     

Regulation of synthesis of ribosomal protein in Neurospora crassa

In Neurospora crassa during a nutritional shift-down transition of growth, the synthesis of rRNA is for about 2 h largely inhibited and the rate of protein synthesis is only partially reduced (by about 25 %). During this period the relative rate of synthesis of individual ribosomal proteins, measured irrespectively of their incorporation into ribosomes, is reduced by 70–80%. The ribosomal proteins synthesized during the shift are stable. Thus, the synthesis of ribosomal proteins appears in N. crassa to be coordinately regulated with that of rRNA.     

Stage-specific changes in protein synthesis during conjugation in Tetrahymena thermophila

Conjugation in the free-living ciliate Tetrahymena thermophila is an inducible developmental system which results in a synchronized reorganization of the genetic material in both mates of a pair. The cytological events were followed by Feulgen stainings of simultaneously mating cells and protein synthesis was revealed using [³⁵S]methionine pulse labelling and two-dimensional gel electrophoresis. At least 33 proteins, including 24 conjugation-specific proteins, with apparent molecular weights (M_r) between 61 and 200 × 10³ are stimulated during conjugation. Two slightly acidic proteins (M_r 89 and 73 × 10³, respectively) are stimulated shortly after mixing of mating-competent cells and mainly before tight pairs are formed. Ten proteins are stimulated during meiosis, and two of these (M_r 90 and 78 × 10³, respectively) are particularly interesting, since they are highly stimulated and more basic (pI values around 8.5) than most other proteins detected. Twelve proteins are stimulated essentially between pairing and early macronuclear development, three are stimulated from shortly before zygote formation and during the postzygotic divisions, and six are stimulated during late conjugation, at various parts of macronuclear development. The functions of the conjugation-stimulated proteins are discussed.     

The effects of protein synthesis inhibitors on theGonyaulax clock

Summary Cycloheximide has been shown to be potent in phase-shifting the circadian glow rhythm of the dinoflagellateGonyaulax polyedra. In experiments in which the cells were exposed to drug pulses of varying concentration (0.35 M to 10 M) and duration (0.5 h to 8 h), the phase shift produced was linear with the log of the product of pulse strength and duration. The sensitivity to drug-induced phase shifting varies as a function of time of day; both advances and delays occurred and, depending on the strength of the perturbation, resulted in strong or weak-type phase response curves. Pulses given at different times after the light/dark to constant dim transition resulted in a crossover from delays to advances at about 15.5 h; this crossover point was the same at 19 °C and 24°C. The occurrence of extended transients following cycloheximide-induced phase advances (but not delays) appears to be the first example of such transients in a microbial circadian system.This research was supported in part by a grant from the National Institutes of Health (GM-19536). J.D. was a NIH predoctoral trainee (T01-GM 00036 and NRSA T32-GM 07598)     

Nuclear protein synthesis in animal and vegetal hemispheres of Xenopus oocytes

Experiments were conducted to determine if nuclear proteins are preferentially synthesized in the vicinity of the nucleus, a factor which could facilitate nucleocytoplasmic exchange. Using Xenopus oocytes, animal and vegetal hemispheres were separated by bisecting the cells in paraffin oil. It was initially established that protein synthesis is not affected by the bisecting procedure. To determine if nuclear protein synthesis is restricted to the animal hemisphere (which contains the nucleus), vegetal halves and enucleated animal halves were injected with [3H]leucine and incubated in oil for 90 min. The labeled cell halves were then fused with unlabeled, nucleated animal hemispheres that had been previously injected with puromycin in amounts sufficient to prevent further protein synthesis. Thus, labeled polypeptides which subsequently entered the nuclei were synthesized before fusion. Three hours after fusion, the nuclei were isolated, run on two-dimensional gels, and fluorographed. Approximately 200 labeled nuclear polypeptides were compared, and only 2 were synthesized in significantly different amounts in the animal and vegetal hemispheres. The results indicate that nuclear protein synthesis is not restricted to the cytoplasm adjacent to the nucleus.     

The effect of mutations in spoOA or spoIIA on the pattern of protein synthesis in Bacillus subtilis under sporulation conditions

Summary By labelling wild-type Bacillus subtilis for 5 min with [³⁵S]-methionine either at the time of resuspension in starvation medium or 1, 2 or 3 h later, and subjecting cell extracts to two-dimensional gel electrophoresis, Yudkin et al. (J. Gen. Microbiol. 1982) detected some 75 proteins whose synthesis started or stopped within the first 3 h of sporulation. Similar experiments have now been done with isogenic strains carrying a spoOA or a spoIIA mutation. The results permit 72 of the changes in protein synthesis to be placed in four classes according to whether they do or do not occur in the mutants as well as in the wild type. The results are in good agreement with the predictions of the dependent-sequence hypothesis, which states that each event that occurs in sporulation depends on the successful completion of all preceding events. The pattern of protein synthesis in the spoIIA mutant differed in some respects from the wild type as early as 1 h after resuspension.     

The isolation of mutants conditionally defective in protein synthesis in Chlamydomonas reinhardi

Summary Canavanine kills Chlamydomonas reinhardi because it is incorporated into protein. This has made it possible to develop a convenient method for isolating mutants which are conditionally defective in protein synthesis. Sixty percent of all mutants isolated by this method prove to have reversible defects in protein synthesis. These mutants have a variety of phenotypes.     

Evidence for aggregation center induction by the ionophore A23187 in the cellular slime mold Polysphondylium violaceum

A thin glass fiber coated with the ionophore A23187 placed among preaggregation amoebae of Polysphondylium violaceum will induce numerous aggregation centers along the fiber. Both calcium and magnesium ions appear to be involved in this induction. A23187 is aiso seen to disrupt the normal cell streaming process by producing secondary centers in the aggregation streams.     

利用红色荧光蛋白分析里氏木霉合成纤维素酶的机理

以红色荧光蛋白作为报告蛋白研究了里氏木霉的纤维素酶合成机理。构建了里氏木霉的表达盒,通过该表达盒使红色荧光蛋白的基因整合到里氏木霉的基因组DNA上,并受纤维二糖水解酶基因启动子的调控,得到重组菌株T.reeseiTR2。在不同的条件下培养T.reeseiTR2,红色荧光蛋白的表达情况可以反映在不同条件下里氏木霉合成纤维素酶的情况。在诱导的情况下,红色荧光蛋白随时间变化的情况与培养液中纤维素酶活性的变化相似,培养至36h后可以观察到荧光,并且不断增强,到菌丝自溶时荧光减弱。另一方面,诱导后里氏木霉菌丝的各个部位均可以观察到荧光,而且分布均匀,表明菌丝的各个部位在纤维素酶合成过程中所起的作用相同。在非诱导的情况下,培养时间较长时也可以观察到较弱的荧光,表明在此条件下里氏木霉仍可以合成少量的纤维素酶,这一结果为解释纤维素诱导里氏木霉合成纤维素酶的机理提供了另一个试验依据。     

Partial inhibition of protein synthesis accelerates the synthesis of porphyrin in heme-deficient mutants of Escherichia coli

Mutants of Escherichia coli defective in the HemA protein grow extremely poorly as the result of heme deficiency. A novel hemA mutant was identified whose rate of growth was dramatically enhanced by addition to the medium of low concentrations of translational inhibitors, such as chloramphenicol and tetracycline. This mutant (H110) carries mutation at position 314 in the hemA gene, which resulted in diminished activity of the encoded protein. Restoration of growth of H110 upon addition of the drugs mentioned above was due to activation of the synthesis of porphyrin. However, this activation was not characteristic exclusively of cells with this mutant hemA gene since it was also observed in a heme-deficient strain bearing the wild-type hemA gene. The activation did not depend on the promoter activity of the hemA gene, as indicated by studies with fusion genes. It appears that partial inhibition of protein synthesis via inhibition of peptidyltransferase can promote the synthesis of porphyrin by providing an increased supply of Guamyl-tRNA for porphyrin synthesis. Glutamyl-tRNA is the common substrate for peptidyltransferase and HemA.     

Protein synthesis in Volvox carteri f. nagariensis

Incorporation of radioactive amino acids was used to examine protein synthesis during the asexual reproductive cycle of the HK10 (female) strain of Volvox carteri f. nagariensis. It was observed that arginine is incorporated into TCA-precipitable material 500 to 1000 times as rapidly as other amino acids. Arginine is taken up by the organisms by a saturable transport system and concentrated against a steep concentration gradient. Intracellular arginine is incorporated with high efficiency: after 1 hr, more than 80% of the intracellular radioactivity is in a TCA-precipitable form. N-terminal analysis ruled out the possibility that this tracer arginine was being incorporated via a ribosome-independent arginyl tRNA-protein transferase and supported the alternative explanation that it was being incorporated via conventional protein synthesis. Proteins labeled with arginine were found to decay with the same time course as those labeled with another amino acid.A method for isolation of somatic and reproductive cells free of cross-contamination is reported. Using this method to isolate cells of prelabeled Volvox, it was observed that arginine is preferentially incorporated into reproductive cells. Reproductive cells exhibited four times as high an arginine-to-lysine incorporation ratio as somatic cells. Over half the total incorporated arginine that was recovered was in reproductive cells. Thus arginine incorporation should serve well as a probe to investigate developmentally significant protein synthesis in the reproductive cells of this species.     

The role of the maturation-promoting factor in controlling protein synthesis in Xenopus oocytes

Oocyte cytosol, containing maturation-promoting factor activity, induces a twofold increase in the rate of protein synthesis as well as inducing germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes. In the current study, it is shown that the cytosol activity responsible for inducing the increase in protein synthesis can be separated from the activity that induces GVBD in recipient oocytes.     

Sphingomyelin synthesis in Ascaridia galli

Adult Ascaridia galli incorporate label from [U-¹⁴C] serine into various intermediates of sphingomyelin synthesis (ketosphinganine, sphinganine, sphingosine, ceramide and sphingomyelin). From the results it is concluded that A. galli possesses the five enzymes involved in sphingomyelin synthesis, namely: serine palmitoyltransferase, 3-ketosphinganine reductase, flavoprotein sphinganine reductase, sphingosine acyltransferase and ceramide choline phosphotransferase.