酵母菌色氨酸合成酶基因的克隆与表达

用RemHI酶切酿酒酵母(Saceharomyces cercuisiae) 1412-4D染色体DNA,通过蔗糖梯度分离2-4kb DNA片段并插入穿棱质粒pCN60,构成1412-4D基因文库。从基因文库中提取重组质粒,转化受体菌C9(a,trp5,adcl,ade6),用直接功能互补法,分离到9株重组质粒,它们都含有3．2kb的TRP5 DNA片段,分别命名为pCN60(trps)1-90转化体中色氨酸合成酶的酶活水平比原始菌株1412-4D高3倍。     

Molecular characterization of TRP1, a gene coding for tryptophan synthetase in the basidiomycete Coprinus cinereus

We utilized a cloned gene (TRP5) encoding tryptophan synthetase (TSase) from Saccharomyces cerevisiae to identify and clone the corresponding gene (TRP1) from the basidiomycete Coprinus cinereus. The primary nucleotide (nt) sequence of this gene was determined and compared to sequences from other filamentous fungi, as well as to other genes coding for TSase. A transformation assay was used to demonstrate that 321 nt, which do not include CAAT or TATAAA elements and precede the translation initiation codon, are sufficient for expression in a variety of chromosomal locations. The coding region (2584 nt) is interrupted at nine positions, and putative splicing signals (5'-GTRNGT...YAG-3') are present in each case. The predicted translation product contains 702 amino acids (aa) and is very similar to other TSases, except in the region of aa 257-296 that connects the alpha and beta functional domains. Both the number and the identity of the aa differ in this region between C. cinereus. S. cerevisiae, and Neurospora crassa. Comparison of exon boundaries in the C. cinereus sequence to the three-dimensional structure of Salmonella typhimurium TSase indicates that there is no simple correlation between exons and major functional domains in this protein.     

Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene

The DNA sequence of a 1.45 kb EcoRI fragment from the yeast (Saccharomyces cerevisiae) TRP1 region has been determined. The fragment contains the TRP1 gene and a yeast chromosomal replicator. The TRP1 gene has been located on the fragment by analysis of potential initiation and termination codons in the DNA sequence. This location has been confirmed by subcloning portions of the fragment. Both the 5' and 3' noncoding regions of the TRP1 gene contain sequence homologies with analogous areas surrounding other yeast genes. The yeast replicator has been localized in a region near the 3' end of the TRP1 gene. The DNA sequence in this region contains several structural features which may be involved in the initiation of DNA replication.     

Control of tryptophan biosynthesis by the methyltryptophan resistance gene in Bacillus subtilis

5-Methyltryptophan-resistant mutants derived from Bacillus subtilis strain 168 synthesize all of the tryptophan biosynthetic enzymes constitutively and excrete tryptophan. These mutants can be divided into three classes: class 1, low enzyme level and low rate of tryptophan excretion; class 2, high enzyme level and intermediate rate of tryptophan excretion; and class 3, high enzyme level and high rate of tryptophan excretion. A bradytrophic requirement for phenylalanine is correlated with the rate of tryptophan excretion. The phenylalanine requirement is relieved when the rate of tryptophan excretion is reduced by either (i) lowering the level of the tryptophan enzymes, (ii) reducing the supply of a tryptophan precursor (chorismate), or (iii) stopping tryptophan synthesis by a mutational block in the pathway. All of the mutants map in a region of the chromosome previously reported as the mtr locus. Our data show that synthesis of the tryptophan enzymes is controlled through the mtr locus but not influenced by precursors of tryptophan.     

The ergosterol biosynthesis inhibitor zaragozic acid promotes vacuolar degradation of the tryptophan permease Tat2p in yeast

Ergosterol is the yeast functional equivalent of cholesterol in mammalian cells. Deletion of the ERG6 gene, which encodes an enzyme catalyzing a late step of ergosterol biosynthesis, impedes targeting of the tryptophan permease Tat2p to the plasma membrane, but does not promote vacuolar degradation. It is unknown whether similar features appear when other steps of ergosterol biogenesis are inhibited. We show herein that the ergosterol biosynthesis inhibitor zaragozic acid (ZA) evoked massive vacuolar degradation of Tat2p, accompanied by a decrease in tryptophan uptake. ZA inhibits squalene synthetase (SQS, EC 2.5.1.21), which catalyzes the first committed step in the formation of cholesterol/ergosterol. The degradation of Tat2p was dependent on the Rsp5p-mediated ubiquitination of Tat2p and was not suppressed by deletions of VPS1, VPS27, VPS45 or PEP12. We will discuss ZA-mediated Tat2p degradation in the context of lipid rafts.     

The ergosterol biosynthesis inhibitor zaragozic acid promotes vacuolar degradation of the tryptophan permease Tat2p in yeast

Ergosterol is the yeast functional equivalent of cholesterol in mammalian cells. Deletion of the ERG6 gene, which encodes an enzyme catalyzing a late step of ergosterol biosynthesis, impedes targeting of the tryptophan permease Tat2p to the plasma membrane, but does not promote vacuolar degradation. It is unknown whether similar features appear when other steps of ergosterol biogenesis are inhibited. We show herein that the ergosterol biosynthesis inhibitor zaragozic acid (ZA) evoked massive vacuolar degradation of Tat2p, accompanied by a decrease in tryptophan uptake. ZA inhibits squalene synthetase (SQS, EC 2.5.1.21), which catalyzes the first committed step in the formation of cholesterol/ergosterol. The degradation of Tat2p was dependent on the Rsp5p-mediated ubiquitination of Tat2p and was not suppressed by deletions of VPS1, VPS27, VPS45 or PEP12. We will discuss ZA-mediated Tat2p degradation in the context of lipid rafts.     

Physiological role of tryptophanase in control of tryptophan biosynthesis in Bacillus alvei

Hoch, J. A. (University of Illinois, Urbana), and R. D. DeMoss. Physiological role of tryptophanase in control of tryptophan biosynthesis in Bacillus alvei. J. Bacteriol. 91:667-672. 1966.-Indole excretion occurred early in the exponential growth phase, and derived mainly from biosynthetic intermediates of tryptophan. Tryptophan cleavage by tryptophanase contributed about 1.5% of the indole excreted. In the presence of exogenous tryptophan (5 to 10 mug/ml), excretion of early indole was not observed. Experiments with isotopically labeled indole and tryptophan showed that a low rate of endogenous tryptophan biosynthesis occurred constantly during growth. Both exogenously and endogenously supplied tryptophan were degraded by tryptophanase. As a consequence, the intracellular tryptophan concentration appeared to be maintained at a constant low level. It was suggested that the action of tryptophanase is an example of an enzymatic mechanism which controls the level of a specific metabolite pool.     

Use of the TRP1 auxotrophic marker for gene disruption and phenotypic analysis in yeast: a note of warning

The TRP1 marker has been commonly used for gene disruption experiments and subsequent phenotypic analysis. However, introduction of the TRP1 gene into a trp1 strain markedly affects growth under many conditions used for phenotypic profiling. Therefore, its use in the past should be revisited and utilization of this marker should be avoided in future analyses.     

The regulation of tryptophan biosynthesis in Pseudomonas aeruginosa

Summary Eighteen auxotrophs of Pseudomonas aeruginosa requiring l-tryptophan for growth were isolated following nitrosoguanidine mutagenesis. Mutant blocks for each step of tryptophan biosynthesis were identified by enzymological assay. A regulatory mutant was characterized which was simultaneously constitutive for the gene products of trpA, trpB and trpD. Another class of regulatory mutant appears to synthesize tryptophan synthetase (i.e., trpE and trpF subunits) constitutively. The results implicate three control entities in the pathway of tryptophan biosynthesis: (i) The gene products of trpA, trpB and trpD are repressible by tryptophan, the range of enzyme specific activity varying at least fifty-fold. (ii) No regulation of the trpC gene product could be demonstrated, indicating that its synthesis is constitutive. (iii) The gene products of rpE and trpF are inducible by indoleglycerol 3-phosphate; the magnitude of induction can exceed 100-fold. These results together with some genetic data indicate a general similarity in gene-enzyme relationships between P. aeruginosa and P. putida. A number of specific differences that distinguish the two species are noted.A mutant blocked in the common pathway of aromatic biosynthesis was used to prove that enzymes of tryptophan biosynthesis other than tryptophan synthetase are not inducible by precursors of the common pathway such as chorismate. It is concluded that the concentration of tryptophan that signals total repression of the gene products of trpA, trpB and trpD is lower than the concentrations necessary for maximal feedback inhibition of anthranilate synthetase and for abolition of the induction of tryptophan synthetase.     

The role of glutathione biosynthesis in heavy metal resistance in the fission yeast Schizosaccharomyces pombe

Abstract: Plants and the fission yeast Schizosaccharomyces pombe synthesize small cadmium-binding peptides, called phytochelatins, in response to cadmium. Derived from glutathione (GSH: λ-Glu-Cys-Gly), they have the general structure (λ-Glu-Cys) n Gly, where n is 2–11. In order to study the biosynthesis of phytochelatins, we used the mutagen N -methyl- N '-nitro- N nitrosoguanidine (MNNG) to select mutants with a lowered GSH content. GSH-deficient mutants show a Cd-sensitive phenotype, whereas resistance to Cu is only slightly influenced. These Cd-sensitive mutants contain 2–15% of the wild-type GSH level. For three mutants a lowered activity of λ-glutamylcysteine synthetase was measured. One of the mutants was transformed to Cd-resistance and the complementing fragment was analyzed further. The complementing fragment hybridized with chromosome III. In the transformants, GSH content was restored up to wild-type levels, whereas the activity of λ-glutamylcysteine synthetase was significantly increased compared with the wild-type. Possible mechanisms for Cd-resistance in the transformants are discussed.     

The role of oxygen in the biosynthesis of cytochrome c oxidase of yeast mitochondria.

The formation of cytochrome c oxidase in yeast is dependent on oxygen. In order to examine the oxygen-dependent formation of the active enzyme, the effect of oxygen on the synthesis and the assembly of cytochrome c oxidase subunits was studied. Pulse-labeling experiments revealed that oxygen has no significant immediate effect on the synthesis of the three mitochondrially made subunits I to III; however, its presence causes subunits I and II to form a complex with the cytoplasmically made subunits VI and VII. This "assembly-inducing" effect can be demonstrated with intact yeast cells as well as with isolated mitochondria. It is independent of cytoplasmic or mitochondrial protein synthesis. After anaerobic growth for 10 or more generations, the intracellular concentrations of individual cytochrome c oxidase subunits drop 10- to 100-fold. Most of these residual subunits are not assembled within a functional cytochrome c oxidase molecule.     

Amplification of the tryptophan operon gene in Escherichia coli chromosome to increase l-tryptophan biosynthesis

Classical mutagenesis could desensitize the feedback inhibition of l-tryptophan (l-Trp) biosynthesis. Among the mutants, a5-fluorotryptophan-resistant strain, Escherichia coli EMS4-C25 produced 3 g/l of l-Trp within 18 h. The feedback-resistant l-Trp operon gene (trp) prepared from E. coli EMS4-C25 was inserted into pUC19 and pHSG576 to generate pTC701 and pTC576, respectively. When pHSG576 and pTC701 were introduced into E. coli EMS4-C25, chromosomal integration occured through homologous recombination. By using Souther hybridization, we demostrated that the integrated plasmids existed as multicopies. The strains with integrated foreign trp operon gene had higher activities of anthranilate synthase and Trp synthase than those found for the host strain and produced 9.2 g/l of l-Trp with 13% conversion yield from d-glucose. The integration and implification of the trp-operon-beraing plasmid avoided the plasmid instability and increased l-TRp production. Correspondence to: E.-C. Chan     

The role of adipose TRP channels in the pathogenesis of obesity

The prevalence of obesity is continuously increasing worldwide. Transient receptor potential (TRP) channels constitute a family of nonselective cation channels that are ubiquitously expressed in mammalian tissues, including adipose tissue. Although TRP channels might be regarded as therapeutic targets for obesity due to the inhibitory effects of their agonists on body weight and adiposity, the exact role of TRP channels in the development of obesity by modulating the function of adipose tissue has not been systemically reviewed. Multiple TRP channels are present in adipocytes and are involved in diverse aspects of cellular function, including differentiation and maturation of white adipose tissue (WAT), browning of WAT and thermogenesis of brown adipose tissue (BAT). Most of these functions are mediated by alterations in intracellular Ca²⁺ levels or subcellular Ca²⁺ signaling pathway. TRP channels influence intracellular Ca²⁺ dynamics through directly mediating Ca²⁺ entry (TRPVs and others) or store-operated mechanisms (TRPCs). Intracellular Ca²⁺ displays a biphasic effect on regulation adipocyte behaviors depending on the differentiation stage, which may account for the different roles of individual TRP channels in regulation of adiposity. This review emphasizes the contribution of TRP channels to obesity and provide an in-depth discussion on the complexity of their mechanism of actions.