Characteristics of Saccharomyces cerevisiae gal1 Delta and gal1 Delta hxk2 Delta mutants expressing recombinant proteins from the GAL promoter

Galactose can be used not only as an inducer of the GAL promoters, but also as a carbon source by Saccharomyces cerevisiae, which makes recombinant fermentation processes that use GAL promoters complicated and expensive. To overcome this problem during the cultivation of the recombinant strain expressing human serum albumin (HSA) from the GAL10 promoter, a gal1 Delta mutant strain was constructed and its induction kinetics investigated. As expected, the gal1 Delta strain did not use galactose, and showed high levels of HSA expression, even at extremely low galactose concentrations (0.05-0.1 g/L). However, the gal1 Delta strain produced much more ethanol, in a complex medium containing glucose, than the GAL1 strain. To improve the physiological properties of the gal1 Delta mutant strain as a host for heterologous protein production, a null mutation of either MIG1 or HXK2 was introduced into the gal1 Delta mutant strain, generating gal1 Delta mig1 Delta and gal1 Delta hxk2 Delta double strains. The gal1 Delta hxk2 Delta strain showed a decreased rate of ethanol synthesis, with an accelerated rate of ethanol consumption, compared to the gal1 Delta strain, whereas the gal1 Delta mig1 Delta strain showed similar patterns to the gal1 Delta strain. Furthermore, the gal1 Delta hxk2 Delta strain secreted much more recombinant proteins (HSA and HSA fusion proteins) than the other strains. The results suggest that the gal1 Delta hxk2 Delta strain would be useful for the large-scale production of heterologous proteins from the GAL10 promoter in S. cerevisiae.     

Hygromycin B-selected cell lines from GAL4-regulated pUAST constructs

Here we report the generation of stable, selectable Drosophila S2 cell lines using the UAS-GAL4 system. Cloning of the hygromycin resistance gene into the pUAST vector and cotransfection with other pUAST constructs in S2 cells results in coexpression of up to four different proteins under hygromycin selection. Protein expression is driven by the ubiquitous Actin5C-GAL4 driver and cell cultures are maintained in hygromycin-supplemented, serum-free media to ensure constitutive protein production. Visual comparison of cells cotransfected with GFP and RFP demonstrates a uniform cell population expressing both markers simultaneously, while Western blot analysis shows concurrent expression of MYC3-tagged proteins. In addition, fluorescent cell sorting (FACS) analysis shows that 80% of the total cell population express the GFP marker. Our data indicate that using this technique it is possible to establish stable, selectable cell lines that provide a pool of readily accessible protein. This facilitates protein-based studies and abolishes the need to carry out time-consuming and expensive transfections.     

A novel genetic tool for clonal analysis of fourth chromosome mutations

The fourth chromosome of Drosophila remains one of the most intractable regions of the fly genome to genetic analysis. The main difficulty posed to the genetic analyses of mutations on this chromosome arises from the fact that it does not undergo meiotic recombination, which makes recombination mapping impossible, and also prevents clonal analysis of mutations, a technique which relies on recombination to introduce the prerequisite recessive markers and FLP-recombinase recognition targets (FRT). Here we introduce a method that overcomes these limitations and allows for the generation of single Minute haplo-4 clones of any fourth chromosome mutant gene in tissues of developing and adult flies.     

Auxin fluxes in the root apex co-regulate gravitropism and lateral root initiation

Root architecture plays an important role in water and nutrient acquisition and in the ability of the plant to adapt to the soil. Lateral root development is the main determinant of the shape of the root system and is controlled by external factors such as nutrient concentration. Here it is shown that lateral root initiation and root gravitropism, two processes that are regulated by auxin, are co-regulated in Arabidopsis. A mathematical model was generated that can predict the effects of gravistimulations on lateral root initiation density and suggests that lateral root initiation is controlled by an inhibitory fields mechanism. Moreover, gene transactivation experiments suggest a mechanism involving a single auxin transport route for both responses. Finally, co-regulation may offer a selective advantage by optimizing soil exploration as supported by a simple quantitative analysis.     

酿酒酵母(Saccharomyces cerevisiae)半乳糖可诱导表达系统特性的研究

把大肠杆菌β-半乳糖苷酶基因克隆到带有酵母半乳糖可诱导启动子GAL1的穿梭表达质粒pYESZ中,并把得到的重组质粒分别转化到两种不同遗传性状的宿主菌中,其中一株菌为蛋白酶活性缺失90％以上的pep4－3突变菌株。通过比较两株重组菌产生的β-半乳糖苷酶活性水平发现在所述实验条件下,蛋白酶缺失突变菌株中产生的β-卜半乳糖苷酶活水平不仅均要高于另一对照菌株,并且pep4-3突变菌株表现出受葡萄糖阻遏的严紧程度高及对诱导反应迅速等特点。此外,带有重组质粒的pep4－3突变菌株在葡萄糖阻遏培养基中最大生长量和重组对照菌株基本相同,但β-半乳糖苷酶在pep4－3突变菌株中的表达对细胞生长的影响明显小于对照菌株。     

High intracellular expression of giant catfish growth hormone under the control of PGK promoter in Saccharomyces cerevisiae

Different yeast plasmid systems containing different promoters such as ADH1, PGK, GAPDH and GAL1, and different selectable markers, such as URA3, TRP1 and leu2-d were compared to obtain the yeast expression system that provides high intracellular expression of giant catfish growth hormone (gcGH). The highest level of gcGH expression was observed in a recombinant yeast under the control of PGK promoter (17.1 mg/l or 1.4 g/0.1 OD). The amount of gcGH was increased six-fold (102.5 mg/l) when cells were grown in a rich medium (YEPD) with the inoculum and medium ratio of 1:1, although the amount of gcGH expression per cell density did not increase (1.0 g/0.1 OD). This indicated that the increased yield of gcGH in rich medium was due to the increased cell density. The aim of the study was to produce high level gcGH in the cells of S. cerevisiae in order to use the yeast cells as potential feed additives to promote growth in giant catfish.     

A genetic screen for elements of the network that regulates neurogenesis in Drosophila

The development of external sensory organs on the notum of Drosophila is promoted by the proneural genes achaete and scute. Their activity defines proneural cell clusters in the wing imaginal disc. Ectopic expression, under control of the GAL4 system, of the proneural gene lethal of scute (l'sc) causes the development of ectopic bristles. Persistent ectopic expression of l'sc is not sufficient to impose a neural fate on any given cell. This implies that mutual inhibition, mediated by the Notch signaling pathway, occurs among the cells of the ectopic proneural cluster. Consequently, the dominant, quantifiable phenotype associated with ectopic expression of l'sc is modified by mutations in genes known to be involved in neurogenesis. This phenotype has been utilized to screen for dominant enhancers and suppressors that modify the number of ectopic bristles. In this way, about 100 000 progeny of EMS or X-ray-treated flies have been analyzed to identify autosomal genes involved in regulation of the neural fate. In addition 1200 chromosomes carrying lethal P-element insertions were screened for modifiers. Besides mutations in genes expected to modify the phenotype, we have isolated mutations in six genes not known so far to be involved in neurogenesis. Received: 20 September 1997 / Accepted: 8 October 1997     

High level expression of isocitrate lyase gene of n-alkane-utilizing yeast Candida tropicalis in Sacchromyces cerevisiae

The genomic DNA of peroxisomal isocitrate lyase (ICL) isolated from an n-alkane-assimilating yeast, Candida tropicalis, was truncated to utilize the original open reading frame under the control of the GAL7 promoter and was expressed in Saccharomyces cerevisiae. The recombinant ICL was synthesized as a functionally active enzyme with a specific activity similar to the enzyme purified from C. tropicalis, and was accounted for approximately 30% of the total extractable proteins in the yeast cells. This recombinant enzyme was easily purified to homogeneity. N-Terminal amino acid sequence, molecular masses of native form and subunit, amino acid composition, peptide maps, and kinetic parameters of the recombinant ICL were essentially the same as those of ICL purified from C. tropicalis. From these facts, S. cerevisiae was suggested to be an excellent microorganism to highly express the genes encoding peroxisomal proteins of C. tropicalis.Abbreviations ICL isocitrate lyase - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis