Characterization of the glyceraldehyde-3-phosphate dehydrogenase gene [correction of glyceraldehyde-3-phosphate gene] and the use of its promoter for heterologous expression in Cryptococcus neoformans, a human pathogen.

The GPD gene encoding glyceraldehyde-3-phosphate dehydrogenase was isolated from Cryptococcus neoformans, a heterobasidiomycetous yeast that is pathogenic to humans. The gene contains 11 introns, differing from the conserved intron positions found in the GPD genes of Basidiomycetes. The predicted amino-acid sequence of this gene is extremely similar to that reported from GPD proteins of other basidiomycetes. The promoter region of the C. neoformans GPD gene was similar to those of other basidiomycetes. Plasmid constructs containing up to 1600 base pairs upstream of the native GPD open reading frame were used to express either the native URA5 gene in a ura5 mutant or the heterologous hphI gene (a bacterial gene that confers resistance to the aminoglycoside hygromycin) in a wild-type strain of C. neoformans. Transformation frequencies resulting from the plasmid-borne Gpdp::URA5 gene were at levels similar to those of the native URA5, which suggested that all the sequences necessary for proper expression were present. Transformation frequencies using the Gpdp::hphI gene constructs were poor. However, addition of DNA sequences flanking the 3'-end of an native C. neoformans gene significantly improved the transformation frequencies resulting from the expression of the heterologous hphI gene.     

Molecular cloning of the promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene that contributes to the construction of a new transformation system in <Emphasis Type="Italic">Coriolus versicolor</Emphasis>

The white-rot basidiomycete Coriolus versicolor secretes several enzymes that participate in the degradation of lignin and various persistent organic pollutants. In this study, we attempted to establish a genetic transformation system with a homogenous promoter sequence for driving the gene for antibiotic resistance. We succeeded in cloning the promoter sequence of the gene for glyceraldehyde-3-phosphate dehydrogenase (gpd), which is expressed at high levels in C. versicolor. The expression vector pT7GPTHPT was constructed, which included a gene for resistance to hygromycin B under control of the gpd promoter. The successful selection of transformants on medium that contained hygromycin B indicated that the system should be useful not only for the genetic transformation of C. versicolor, but also for the overproduction of useful fungal enzymes such as laccase and peroxidase.     

Tissue- and cell-specific expression of human sn-glycerol-3-phosphate dehydrogenase in transgenic mice.

Comparison of the promoter sequence for the sn-glycerol-3-phosphate dehydrogenase (GPDH, EC 1.1.1.8) genes in mice and humans showed that there were three promoter domains conserved in evolution (1). To study the functional organization of the GPDH promoter, we generated transgenic mice carrying the complete human gene, GPD1. The level of human and mouse GPDH activity was measured in each tissue and the amount of human-mouse GPDH heterodimer was used as a sensitive indicator of cell-specific expression of GPD1. During postnatal development and in adult tissues of the transgenic mice, human GPDH was expressed at levels that corresponded closely to the expression of the endogenous mouse gene, Gdc-1. Surprisingly, deletion of the evolutionarily conserved fat-specific elements (FSE) in the proximal promoter region failed to reveal any alterations in GPD1 expression that were specific for either white or brown adipose tissue.     

Usefulness of heterologous promoters in the Pseudozyma flocculosa gene expression system

The basidiomycetous fungus Pseudozyma flocculosa represents a promising new host for the expression of complex recombinant proteins. Two novel heterologous promoter sequences, the Ustilago maydis glyceraldehyde-3-phosphate dehydrogenase (GPD) and Pseudozyma tsukubaensis alpha-glucosidase promoters, were tested for their ability to provide expression in P. flocculosa. In liquid medium, these two promoters produced lower levels of intracellular green fluorescent protein (GFP) as compared to the U. maydis hsp70 promoter. However, GPD and alpha-glucosidase sequences behaved as constitutive promoters whereas the hsp70 promoter appeared to be morphology-dependent. When using the hsp70 promoter, the expression of GFP increased proportionally to the concentration of hygromycin in the culture medium, indicating possible induction of the promoter by the antibiotic. Optimal solid-state culture conditions were designed for high throughput screening of hygromycin-resistant transformants with the hsp70 promoter in P. flocculosa.     

CCAAT/enhancer binding protein-beta negatively regulates the expression of glycerol-3-phosphate dehydrogenase 1 in pig PK-15 cells

Soluble glycerol-3-phosphate dehydrogenase 1 (GPD1, EC 1.1.1.8) plays important roles in the synthesis of triacylglycerol and in the glycerol-3-phosphate shutter. Though GPD1 is expressed in most adult tissues, little is known about the regulation of its expression. In this study, we analyzed the characters, organization and core region of the promoter of pig GPD1 gene by in silico analysis and activity detection of deletion mutants. We also identified and testified the negative regulation effect of C/EBP β on pig GPD1 gene by Chromatin immunoprecipitation (ChIP) assay and over-expression experiments in cultured pig kidney cells. Compared to that of human, pig GPD1 gene promoter has three conserved regions and one deletion region. In silico analysis indicated that pig GPD1 promoter was TATA-less with at least 3 CpG islands of over 200 bp in length and over 60% in GC content. The activity detection of deletion mutants suggested that the essential elements required for the optimal promoter activity scatter in the promoter region, while the core promoter region was from -422 bp to -1 bp. Chromatin immunoprecipitation (ChIP) assay results indicated that C/EBP β had plenty of binding sites in pig GPD1 promoter with the common cis-element (5’- TKNNGCAAK -3’). The over-expression examination of C/EBP β showed that the expression of GPD1 was negatively regulated by C/EBP β in pig kidney cells. Overall, our study revealed that the pig GPD1 promoter is a TATA-less promoter, and in promoter region, the binding sites of C/EBP β share common motif of (5’-TKNNGCAAK -3’). We also showed that pig GPD1 gene is regulated negatively by C/EBP β in cultured kidney cells.     

Improvement of the transformation efficiency of Flammulina velutipes Fv-1 using the glyceraldehyde-3-phosphate dehydrogenase gene promoter

To improve the expression level of heterologous genes in Flammulina velutipes Fv-1, we constructed new vectors having glyceraldehydes-3-phosphate dehydrogenase (gpd) gene promoter to control the expression of target genes. When the hygromycin B phosphotransferase (hph) gene from Escherichia coli was controlled by the gpd promoter, transformation efficiency was 3-fold higher than the case of that controlled by the tryptophan synthetase gene (trp1) promoter.     

Sequence conservation and structural organization of the glycerol-3-phosphate dehydrogenase promoter in mice and humans.

Cloned segments of the mouse glycerol-3-phosphate dehydrogenase (GPDH) gene, Gdc-1, were used to screen a human library. Human clones obtained spanned 25 kilobases of genomic DNA containing the human GPDH gene, GPD1. The 4 kb of sequence obtained from the 5'-flanking region and first exon of GPD1 was compared with the corresponding mouse sequence. Both sequences share a HindIII site located in what has proven to be the highly conserved 3' untranslated region of an upstream gene of unknown function, D15Kzl. The 3.6-kilobase segment of mouse DNA located between D15Kzl and Gdc-1 was provisionally termed the GPDH promoter. Alignment of the mouse promoter with the corresponding human sequence revealed two conserved domains. An upstream distal promoter region is approximately 900 base pairs in length. A downstream or proximal promoter region consists of approximately 300 base pairs immediately upstream of a TATA-like box and contains the fat-specific elements 1 and 2. Analysis of the chromatin structure of the Gdc-1 promoter revealed four DNase I-hypersensitive sites. They were present in DNA of liver and brown fat, in which GPDH expression is high, but were absent in DNA of spleen, in which GPDH expression is low. Methylation studies of the promoter showed it to be heavily methylated in sperm. However, the DNA from each adult somatic tissue had a unique distribution of nonmethylated sites and could easily be identified by its methylation pattern. These data suggest a structural model of the promoter that explains how Gdc-1 expression is differentially regulated in many types of cells.     

产甘油假丝酵母胞浆3-磷酸甘油脱氢酶基因（CgGPD）功能分析

【目的】从高产甘油生产菌株产甘油假丝酵母（Candida glycerinogenes）基因组中克隆了NAD+依赖3-磷酸甘油脱氢酶编码基因（CgGPD）,但是该基因及其上游调控序列具体的功能还是未知的。本文研究了CgGPD基因及其上游调控序列的功能。【方法】本文以酿酒酵母（Saccharomyces cerevisiae）及其渗透压敏感型突变株为宿主,构建3种不同的酵母表达载体导入酵母细胞,研究了不同酵母转化子在渗透压胁迫条件下CgGPD基因表达对细胞的耐高渗透压胁迫应答及其细胞的甘油合成能力的影响。【结果】实验结果表明无论是以来源于S. cerevisiae 的TPI启动子还是来源于CgGPD基因的启动子,过量表达CgGPD基因的转化子均能够显著加速葡萄糖消耗速度和提高甘油合成能力,在gpd1/gpd2突变株中表达CgGPD基因能够消除细胞对外界高渗透压的敏感性,同时转化子胞内甘油大量积累。【结论】CgGPD基因在野生型酵母S. cerevisiae W303-1A表达显著提高细胞的甘油合成能力,在gpd/1gpd2突变株中能够互补GPD1基因的功能,CgGPD基因表达受渗透压诱导 调控。     

A transformation system for an ectomycorrhizal basidiomycete, Lyophyllum shimeji

A transformation vector, pLS-hph, was constructed with the promoter and terminator of the glyceraidehyde-3-phosphate dehydrogenase (GPD) gene derived from an ectomycorrhizal basidiomycete, Lyophyllum shimeji, and with the hygromycin B (HmB) phosphotransferase (hph) gene from Escherichia coli. This vector was introduced into protoplasts of L. shimeji and 3.4 transformants per microg plasmid DNA were obtained. In most of the transformants, multiple copies of the vector were integrated into the genomic DNA. The results indicate that pLS-hph is a useful vector for L. shimeji.     

CaMV 35S promoter directs β-glucuronidase expression in Ganoderma lucidum and Pleurotus citrinopileatus

The cauliflower mosaic virus (CaMV) 35S promoter has been most commonly used in plant transformation studies, but its activity in mushrooms has not been reported. p301-b is a binary vector containing a bialaphos resistance gene driven by the promoter of Lentinus edodes glyceraldehyde-3-phosphate dehydrogenase (GPD) gene. CaMV 35S-GUS was inserted into p301-b, and the resulting construct p301-bG was transformed to protoplasts of Ganoderma lucidum and basidiospores of Pleurotus citrinopileatus. GUS activity was observed in the transformants, indicating that CaMV 35S promoter can direct expression of exogenous gene in the mushrooms. This is the first report on the application of CaMV 35S promoter in genetic modification of mushrooms.     

Cloning and sequence analysis of Mucor circinelloides glyceraldehyde-3-phosphate dehydrogenase gene

A genomic library of Mucor circinelloides ATCC 1216b has been constructed in Lambda Fix II vector. The library has an average insert site of 10 kb and covers the genome 12 times. The M. circinelloides gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by PCR reaction. The complete nucleotide sequence encodes a putative polypeptide chain of 339 amino acids interrupted by 3 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the GPD proteins from other filamentous fungi. The promoter region, containing a consensus TATA and CAAT box and a 298 nucleotid long termination region were also determined.     

Development of a simple and efficient transformation system for the basidiomycetous medicinal fungus Ganoderma lucidum

In this study, we report the development of a simple and efficient system for genetic transformation of the medicinal fungus Ganoderma lucidum. Various parameters were optimized to obtain successful Agrobacterium tumefaciens-mediated transformation. Co-cultivation of bacteria and protoplast at a ratio of 1,000:1 at 25°C in medium containing 0.2 mM acetosyringone was found to be the optimum condition for high efficiency transformation. Four plasmids, each carrying a different promoter driving the expression of an antibiotic resistance marker, were tested. The construct carrying the Ganoderma lucidum glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter showed good transformation efficiency, whereas constructs with the GPD promoter from ascomycetes were ineffective. Our analysis showed that over 70% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. We were able to detect the expression of EGFP and GUS reporter genes in the Ganoderma lucidum transformants by fluorescence imaging and histochemical staining assays respectively. Our results demonstrate a new transgenic approach that will facilitate Ganoderma lucidum research.     

产甘油假丝酵母与酿酒酵母胞浆3-磷酸甘油脱氢酶基因的功能比较

胞浆3-磷酸甘油脱氢酶(GPD)是酿酒酵母细胞甘油合成过程中的关键限速酶．尽管高产甘油菌株产甘油假丝酵母基因组中编码该酶的基因CgGPD已经被克隆出来,但是具体的功能,特别是与酿酒酵母GPD1和GPD2基因的功能比较值得进一步研究．以酿酒酵母渗透压敏感型的gpd1/gpd2和gpd1突变株为宿主,分别导入CgGPD、GPD1和GPD2基因,比较分析了CgGPD、GPD1和GPD2基因在高渗透压胁迫条件下和厌氧环境中的表达调控,及其对细胞甘油合成能力的影响．研究发现,GPD1基因受到渗透压诱导表达,GPD2基因在细胞厌氧条件下起着氧化还原平衡调节作用,而CgGPD基因不仅能够在渗透压胁迫条件下通过过量快速合成甘油调节渗透压平衡,而且能够在厌氧培养环境中互补GPD2基因的缺失,使gpd1/gpd2缺失突变株能够正常生长,同时提高了突变株的甘油合成能力．结果表明,CgGPD基因在gpd1/gpd2缺失突变株中既具有GPD1基因的功能,又能发挥GPD2基因的功能．