GFP工程酵母菌的构建及其对Cu2+的荧光响应

从酿酒酵母基因组DNA中克隆到金属硫蛋白启动子(PCUP1)片段,将绿色荧光蛋白(GFP)基因置于PCUP1的调控下,构建重组质粒pCUP9K-GFP,并通过氯化锂法转化毕赤酵母,获得工程菌株。工程菌细胞及其发酵液中可检出GFP荧光,表明PCUP1能启动外源基因GFP转录,使工程菌表达并分泌GFP。研究发现,工程菌培养液中分别加入10μmol/L的铜、铬、镉和砷离子后,铜处理组GFP荧光强度明显增加,其余三种离子对工程菌荧光强度影响不大;用铜离子诱导后,工程菌发酵上清液的荧光强度明显增强,并与铜离子浓度(0～1mmol/L)呈正相关。研究表明,该工程菌中启动子PCUP1受铜离子诱导,GFP的表达对铜离子具有剂量依赖性,在一定浓度范围内,GFP荧光强度与铜离子浓度呈正相关。     

Efficient expression of the yeast metallothionein gene in Escherichia coli.

The yeast metallothionein gene CUP1 was cloned into a bacterial expression system to achieve efficient, controlled expression of the stable, unprocessed protein product. The Escherichia coli-synthesized yeast metallothionein bound copper, cadmium, and zinc, indicating that the protein was functional. Furthermore, E. coli cells expressing CUP1 acquired a new, inducible ability to selectively sequester heavy metal ions from the growth medium.     

The CUP2 gene product regulates the expression of the CUP1 gene, coding for yeast metallothionein.

The yeast CUP1 gene codes for a copper-binding protein similar to metallothionein. Copper sensitive cup1s strains contain a single copy of the CUP1 locus. Resistant strains (CUP1r) carry 12 or more multiple tandem copies. We isolated 12 ethyl methane sulfonate-induced copper sensitive mutants in a wild-type CUP1r parental strain, X2180-1A. Most mutants reduce the copper resistance phenotype only slightly. However, the mutant cup2 lowers resistance by nearly two orders of magnitude. We cloned CUP2 by molecular complementation. The smallest subcloned fragment conferring function was approximately 2.1 kb. We show that CUP2, which is on chromosome VII, codes for or controls the synthesis or activity of a protein which binds the upstream control region of the CUP1 gene on chromosome VIII. Mutant cup2 cells produced extremely low levels of CUP1-specific mRNA, with or without added copper ions and lacked a factor which binds to the CUP1 promoter. Integrated at the cup2 site, the CUP2 plasmid restored the basal level and inducibility of CUP1 expression and led to reappearance of the CUP1-promoter binding factor. Taken collectively, our data establish CUP2 as a regulatory gene for expression of the CUP1 metallothionein gene product.     

Versatile cassettes designed for the copper inducible expression of proteins in yeast

A series of yeast expression vectors and cassettes utilizing the CUP1 gene of Saccharomyces cerevisiae have been constructed. The cassettes contain multiple cloning sites for gene fusions and were created by inserting a 27-bp polylinker at the +14 position of the CUP1 gene. The cassettes are portable as restriction fragments and enable copper-regulated expression of foreign proteins in S. cerevisiae. In copper sensitive yeast, multiple copies of the CUP1 cassettes confer copper resistance due to the production of the copper metallothionein. Genes cloned into the CUP1 cassettes, however, usually prevent translation of the metallothionein leading to a loss of resistance. This could be useful for one-step cloning into yeast.     

Transgenic Mice Expressing Yeast CUP1 Exhibit Increased Copper Utilization from Feeds

Copper is required for structural and catalytic properties of a variety of enzymes participating in many vital biological processes for growth and development. Feeds provide most of the copper as an essential micronutrient consumed by animals, but inorganic copper could not be utilized effectively. In the present study, we aimed to develop transgenic mouse models to test if copper utilization will be increased by providing the animals with an exogenous gene for generation of copper chelatin in saliva. Considering that the S. cerevisiae CUP1 gene encodes a Cys-rich protein that can bind copper as specifically as copper chelatin in yeast, we therefore constructed a transgene plasmid containing the CUP1 gene regulated for specific expression in the salivary glands by a promoter of gene coding pig parotid secretory protein. Transgenic CUP1 was highly expressed in the parotid and submandibular salivary glands and secreted in saliva as a 9-kDa copper-chelating protein. Expression of salivary copper-chelating proteins reduced fecal copper contents by 21.61% and increased body-weight by 12.97%, suggesting that chelating proteins improve the utilization and absorbed efficacy of copper. No negative effects on the health of the transgenic mice were found by blood biochemistry and histology analysis. These results demonstrate that the introduction of the salivary CUP1 transgene into animals offers a possible approach to increase the utilization efficiency of copper and decrease the fecal copper contents.     

Regulation of the yeast metallothionein gene

To study regulation of the yeast CUP1 gene, we have employed plasmids containing the CUP1 regulatory sequences fused to the Escherichia coli galK gene. A comparison of galK expression from low- and high-copy-number CUP1/galK fusion plasmids demonstrated that both basal and induced levels of galactokinase (GalK) increase proportionately with plasmid copy number. Host strains with an amplified, single or deleted CUP1 locus were compared to look for effects of chromosomal CUP1 gene dosage on expression from the episomal CUP1 promoter. Basal GalK levels are similar in CUP1R and cupls hosts, but can be induced to higher levels in the cup1s than the CUP1R host. In contrast, in a strain deleted for the chromosomal copy of CUP1, synthesis of GalK is constitutive but can be induced to yet higher levels by copper. A hybrid vector, placing the CUP1 coding sequence under the control of a constitutive promoter, was constructed. Introduction of this hybrid CUP1 gene into the deletion host containing the CUP1/galK plasmid restores regulation. Thus, metallothionein, in trans, can effect repression of the CUP1 promoter. The possible roles of metallothionein and free copper in CUP1 regulation are discussed.     

Industrial yeasts display tandem gene iteration at the CUP1 region.

The gene copy number at the CUP1 locus and the resistance level to external copper was directly correlated in five wild-type commercial Saccharomyces strains. An increased copy number of the CUP1 gene leads to increased accumulation of chelatin mRNA, which codes for a low-molecular-weight, copper-binding protein. The enhanced production of this rapidly inducible protein mediates resistance of the cell to copper. Industrial yeasts exhibit homologies to the amplified copper resistance repeat unit found in laboratory strains. However, the extent of tandem iteration is strain dependent, and the repetitious unit is either 1.7 or 1.5 kilobases in length compared with the 2.0-kilobase unit in laboratory strains. Strain 522 (Montrachet) contains two chromosome VIII segments distinguishable by their numbers of repeat units (2 and 11) and the size of the units (1.5 and 1.7 kilobases). Distillers yeast 513 carries a 1.5-kilobase repeat unit on each homologous chromosome, although they contain nine and five iterations, respectively.     

Mutational Analysis of Pre-mRNA Splicing in Saccharomyces Cerevisiae Using a Sensitive New Reporter Gene, Cup1

We have developed a new reporter gene fusion to monitor mRNA splicing in yeast. An intron-containing fragment from the Saccharomyces cerevisiae ACT1 gene has been fused to CUP1, the yeast metallothionein homolog. CUP1 is a nonessential gene that allows cells to grow in the presence of copper in a dosage-dependent manner. By inserting previously characterized intron mutations into the fusion construct, we have established that the efficiency of splicing correlates with the level of copper resistance of these strains. A highly sensitive assay for 5' splice site usage was designed by engineering an ACT1-CUP1 construct with duplicated 5' splice sites; mutations were introduced into the upstream splice site in order to evaluate the roles of these highly conserved nucleotides in intron recognition. Almost all mutations in the intron portion of the 5' consensus sequence abolish recognition of the mutated site, while mutations in the exon portion of the consensus sequence have variable affects on cleavage at the mutated site. Interestingly, mutations at intron position 4 demonstrate that this nucleotide plays a role in 5' splice site recognition other than by base pairing with U1 snRNA. The use of CUP1 as a reporter gene may be generally applicable for monitoring cellular processes in yeast.     

Structural and functional studies of the amino terminus of yeast metallothionein

Purified yeast copper-metallothionein lacks 8 amino-terminal residues that are predicted from the DNA sequence of its gene. The removed sequence is unusual for metallothionein in its high content of hydrophobic and aromatic residues and its similarity to mitochondrial leader sequences. To study the significance of this amino-terminal cleavage, several mutations were introduced into the metallothionein coding gene, CUP1. One mutant, which deletes amino acid residues 2-8, had a minor effect on the ability of the molecule to confer copper resistance to yeast but did not affect CUP1 gene regulation. A second mutation, which changes two amino acids adjacent to the cleavage site, blocked removal of the extension peptide but had no effect on copper detoxification or gene regulation. Immunofluorescence studies showed that both the wild-type and these two mutant proteins are predominantly cytoplasmic with no evidence for mitochondrial localization. The cleavage site mutation allowed isolation and structural characterization of a full length metallothionein polypeptide. The copper content and luminescent properties of this molecule were identical to those of the truncated wild-type protein indicating a homologous cluster structure. Moreover, the amino-terminal peptide was selectively removed by various endopeptidases and an exopeptidase suggesting that it does not participate in the tertiary fold. These results argue that the amino-terminal peptide is not required for either the structural integrity or biological function of yeast metallothionein.