The role of ALA-S and ALA-D in regulating porphyrin biosynthesis in a normal and a HEM R+ mutant strain of Saccharomyces cerevisiae

Catabolite repression and derepression on δ-aminolevulinate synthase (ALA-S) and δ-aminolevulinate dehydratase (ALA-D) in a normal yeast strain, D27, and its derived D27/C6 (HEM R⁺) were investigated. ALA-S and ALA-D activities and intracellular ALA (I-ALA) at different physiological states of the cells were measured. In YPD medium, under conditions of repression and when glucose was exhausted, both strains behaved identically as if the mutation was not expressed. In YPEt medium, however, both ALA-S and ALA-D activities were higher than in YPD, but the I-ALA content and the enzymic activity profiles shown by the two strains were quite different. It appears, therefore, that the mutation causes a deregulation of ALA-S, so that its activity is kept at a high level throughout the cell cycle. This would explain the increased levels of cytochromes present in the mutant. This mutation may affect some regulatory aspect of ALA formation and renders an ALA-S of high activity; moreover, this enzyme species seems to be more stable than in the normal strain.     

Assembly of phosphofructokinase-1 from Saccharomyces cerevisiae in extracts of single-deletion mutants

Phosphofructokinase-1 from Saccharomyces cerevisiae is an octameric enzyme comprising two non-identical subunits, α and β, which are encoded by the unlinked genes PFK1 and PFK2. In this paper, assembly and reactivation of the enzyme have been studied in cell-free extracts of single-deletion mutants. In contrast to the previously described lack of phosphofructokinase-1 activity in cell-free extracts of these mutants, we could measure a temporary enzyme activity immediately after lysis of protoplasts. This result supports the assumption that each of the subunits forms an enzyme structure which is active in vivo but not stable after cell disruption. Upon mixing of separately prepared cell-free extracts of both deletion mutants very low activity could be measured. About 40% of the wild-type activity was regained when both mutants were mixed prior to disruption. The reactivation rate could be slightly increased by addition of ATP and fructose 6-phosphate and was found to be a function of the growth state, particularly of the β-subunit-carrying cells. The individual subunits did not interact with Cibacron Blue F3G-A, a biomimetic ligand of phosphofructokinase-1. After reassembly of both subunits in vitro a strong affinity of the reconstituted phosphofructokinase-1 to the dye-ligand was observed. The inability of the subunits to reconstitute under certain conditions seems to result from alterations of the intracellular environment following disruption. These changes give rise to induce an unproductive side reaction like self-aggregation of the subunits. Because reconstitution of phosphofructokinase-1 from S. cerevisiae behaves in a similar way to that of hemoglobin and luciferase, we would speculate a general mechanism for assembly of oligomeric proteins in vivo. © 1998 John Wiley & Sons, Ltd.     

A Conditional Sterol Esterification Defect in Yeast having either a sec1 or sec5 Mutation in the Secretory Pathway

Two temperature-conditional secretory mutations, sec1 and sec5, cause the accumulation of post-Golgi vesicles when strains containing these mutations are grown at 37°C. In addition to accumulating vesicles, the mutants do not esterify free sterol on rich media at the restrictive temperature. It is the high level of inositol in the media that causes this condition in the yeast Saccharomyces cerevisiae, not a defective steryl ester synthase or lack of substrates. When strains containing the sec1 or sec5 mutation were transformed separately with a plasmid carrying SEC1 and SEC5, the esterification and secretory defects were alleviated. Double mutants containing sec6, sec14 or sec18 with either a sec1 or sec5 mutation have normal esterification levels. Strains with suppressor mutations were isolated that grew at 37°C, esterified sterols and had diminished accumulation of vesicles, when grown at the restrictive temperature on defined media with additional inositol. Electron microscopy was used to examine vesicle accumulation, the number of lipid droplets, and to further characterize the esterification defect. When grown at 37°C on defined medium, the strains with sec5 or sec1 accumulated the usual secretory vesicles, but when grown under similar conditions with elevated levels of inositol, accumulated an additional vesicular-like body. © 1997 John Wiley & Sons, Ltd.     

高产柠檬酸的SHAM特性黑曲霉突变株的选育

研究了SHAM (水杨酸氧肟酸 )对产酸的影响。 5mg/ 6 0mL的SHAM基本抑制柠檬酸的产生。当W 3菌株在加有 10mg/ 10mLSHAM(L1)的平板上生长时 ,只能着生 10 %的孢子 ,在加有 2 0mg/ 10mLSHAM (L2 )的平板上生长时不能着生孢子。据此选育出不能够在L1平板上着生孢子的SHAM敏感株和在L2平板上能够着生孢子的SHAM抗性株。通过发酵试验 ,SHAMS- 2菌株产柠檬酸比出发菌株W 3高 4 0 % ,转化率达到 91 7%     

Expression of functional papain precursor in Saccharomyces cerevisiae: rapid screening of mutants

A microbial expression system for the study of the cysteineprotease papain has been developed as a more useful alternativeto the insect cell/baculovirus expression system we have previouslyused. A synthetic papain precursor (propapain) gene was expressedin the yeast Saccharomyces cerevisiae under the control of the-factor promoter. Efficient expression required fusion of thepropapain sequence with the yeast -factor prepro region anda yeast host cell defective in the synthesis of vacuolar proteases.Surprisingly, the glycosylated form of the inactive papain precursoris not secreted, but accumulates within the yeast cell. Completeconversion of the intracellular zymogen into active mature papaincould be achieved in vitro. Purified recombinant papain producedby the yeast system has kinetic characteristics similar to thoseof the natural enzyme. An advantage of the yeast expressionsystem over the baculovirus/insect cell system is that we canperform mutagenesis and screening of papain mutants very efficiently.We have set up a ‘one-tube’ screening procedurefor the simultaneous characterization of numerous mutants ofthe papain precursor. Yeast cells are grown and lysed in microtiterplate wells and the released papain precursor is then activatedto mature papain. This assay allows easy discrimination betweenproteins with close to wild type properties and proteins thatare not functional. We have applied this assay to investigatethe spectrum of amino acids which are tolerated at Asnl75 ofpapain using two independently derived libraries of mutantsat this position. Many amino acid substitutions at this positionare not accepted; only the reintroduction of Asn restored normalfunction.     

The carboxyl-terminal region of human interferon {gamma} is important for biological activity: mutagenic and NMR analysis

Deletion of nine amino acids from the carboxyl terminus of humanIFN (residues 138–146; LFRGRRASQ) resulted in a 7-foldincrease in specific antiviral activity. Similar increases inreceptor binding affinity were seen. Deletion of residues 136and 137 (QM) had little additional effect, but removal of Ser135resulted in a sharp drop in antiviral activity. Further removalof residues 133 and 134 (KR) lowered antiviral activity to 1%of the peak value. Comparison of the proton NMR spectra of selecteddeletions down to residue 132 showed that there was no significantchange in the core protein structure. Deletions down to residue125 had the same antiviral activity as those to 132, but changescould now be seen in the aromatic proton NMR spectrum of thisshorter derivative. Substitution of the homologous murine sequencebetween residues 124 and 130 (human SPAAKTG; murine LPESSLR)resulted in only a small decrease in antiviral activity, furthersuggesting that the precise sequence in this region was notcritical for activity. Ser135 was substituted with a numberof other amino acids with little or no change in activity. Theimportance of the residues between 131 and 134 for biologicalactivity was corroborated by mutagenesis, although some substitutionsin this region were tolerated.     

植物乳杆菌噬菌体Lpla的分离鉴定及抗噬菌体菌株的筛选

从泡菜中分离得到1 株以植物乳杆菌WCFS1为宿主菌的烈性噬菌体,命名为Lpla。一步生长曲线结果表明Lpla的潜伏期为15 min,裂解期为180 min,裂解量为43 PFU/cell。根据透射电镜观察的形态将其归为长尾病毒科,B1类。噬菌体的理化性质结果表明,Lpla对酸、碱的耐受性较强,对乙醇、温度和紫外射线的耐受性较低。吸附实验结果显示,Lpla在4 ℃时吸附率最大。Ca2＋、Mg2＋均可促进Lpla对宿主菌的吸附,热灭活宿主菌可使Lpla的吸附率下降20%。本研究筛选出6 株对噬菌体Lpla具有稳定抗性的植物乳杆菌,为制定出有效的噬菌体防治策略提供理论依据,并为进一步将抗性菌株应用于生产实践奠定基础。     

Classical transketolase functions as the formaldehyde-assimilating enzyme during growth of a dihydroxyacetone synthase-negative mutant of the methylotrophic yeast Hansenula polymorpha on mixtures of xylose and methanol in continuous cultures

Contrary to expectation, a mutant of Hansenula polymorpha blocked in dihydroxyacetone (DHA) synthase was able to assimilate methanol-carbon when grown in chemostat culture on mixtures of xylose and methanol. Incubation of a DHA synthase- and DHA kinase-negative double mutant resulted in DHA accumulation, indicating that a DHA synthase-type of reaction was involved. Low residual DHA synthase activity subsequently was shown to be present when using an assay with improved sensitivity. This activity was not associated with the (mutated) DHA synthase protein, which was still present in the peroxisomes, but with the enzyme transketolase. Transketolase from methanol grown cells was purified (525-fold) to homogeneity in 9% yield. The native enzyme was dimeric, as has been reported fro other transketolases, with a subunit molecular weight of 74000. The affinity of the purified enzyme for formaldehyde was low (K_m = 5 mM ), but high for xylulose-5-phosphate (ca. 10 μM ). The in vivo functioning of transketolase in formaldehyde assimilation, and the influence of the hydration state of formaldehyde is discussed.     

A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white

To study cell wall assembly, a simple screening method was devised for isolating cell wall mutants. Mutagenized cells were screened for hypersensitivity to Calcofluor White, which interferes with cell wall assembly. The rationale is that Calcofluor White amplifies the effect of cell wall mutations. As a result, the cells stop growing at lower concentrations of Calcofluor White than cells with normal cell wall. In this way, 63 Calcofluor White-hypersensitive (cwh), monogenic mutants were obtained, ordered into 53 complementation groups. The mannose/glucose ratios of the mutant cell walls varied from 0.15 to 3.95, while wild-type cell walls contained about equal amounts of mannose and glucose. This indicates that both low-mannose and low-glucose cell wall mutants had been obtained. Further characterization showed the presence of three low-mannose cell wall mutants with a mnn9-like phenotype, affected, however, in different genes. In addition, four new killer-resistant (kre) mutants were found, which are presumably affected in the synthesis of β1,6-glucan. Most low-glucose cell wall mutants were not killer resistant, indicating that they might be defective in the synthesis of β1,3-glucan. Eleven cwh mutants were found to be hypersensitive to papulacandin B, which is known to interfere with β1,3-glucan synthesis, and four cwh mutants were temperature-sensitive and lysed at the restrictive temperature. Finally, nine cwh mutants were hypersensitive to caffeine, suggesting that these were affected in signal transduction related to cell wall assembly.     

LysM Receptor-Like Kinase LYK9 of Pisum Sativum L. May Regulate Plant Responses to Chitooligosaccharides Differing in Structure

This study focused on the interactions of pea (Pisum sativum L.) plants with phytopathogenic and beneficial fungi. Here, we examined whether the lysin-motif (LysM) receptor-like kinase PsLYK9 is directly involved in the perception of long- and short-chain chitooligosaccharides (COs) released after hydrolysis of the cell walls of phytopathogenic fungi and identified in arbuscular mycorrhizal (AM) fungal exudates. The identification and analysis of pea mutants impaired in the lyk9 gene confirmed the involvement of PsLYK9 in symbiosis development with AM fungi. Additionally, PsLYK9 regulated the immune response and resistance to phytopathogenic fungi, suggesting its bifunctional role. The existence of co-receptors may provide explanations for the potential dual role of PsLYK9 in the regulation of interactions with pathogenic and AM fungi. Co-immunoprecipitation assay revealed that PsLYK9 and two proposed co-receptors, PsLYR4 and PsLYR3, can form complexes. Analysis of binding capacity showed that PsLYK9 and PsLYR4, synthesized as extracellular domains in insect cells, were able to bind the deacetylated (DA) oligomers CO5-DA–CO8-DA. Our results suggest that the receptor complex consisting of PsLYK9 and PsLYR4 can trigger a signal pathway that stimulates the immune response in peas. However, PsLYR3 seems not to be involved in the perception of CO4-5, as a possible co-receptor of PsLYK9.