Expression and in vivo determination of firefly luciferase as gene reporter in Saccharomyces cerevisiae

The LUC gene coding for Photinus pyralis firefly luciferase was cloned in different yeast episomal plasmids in order to assess its possibilities as an in vivo reporter gene. Activity of the enzyme in transformed cells in vivo was measured by following light emission and assay conditions optimized in intact cells, with regard to oxygen concentration, temperature, cell concentration in assay mixtures and external ATP concentration. Among the factors tested, light emission was drastically influenced by the external pH in the assay (which resulted in a ten-fold amplification signal) and by substrate permeability. The growth phase of the cells was also important for the level of activity detected. Cloning of firefly luciferase gene under the control of different yeast-regulated promoters (ADH1, GAL1–10) enabled us to measure their strength which correlated well with previously described data. We conclude that firefly luciferase is an adequate gene reporter for the in vivo sensitive determination of gene expression and promoter strength in yeast.     

One-step measurement of firefly luciferase activity in yeast

Firefly luciferase is often used as a sensitive genetic reporter in various cell types. The pitfall in yeast, however, has been the need to break down the rigid cells in order to measure the enzyme activity. In this study we have removed the peroxisomal targeting codons from the Photinus pyralis luciferase gene (luc) and shown that in the yeast Saccharomyces cerevisiae this modified luciferase gives high levels of light emission that is easy to measure from intact living cells. Furthermore, cells with the modified luciferase grew essentially faster than those with the wild-type luciferase, indicating that peroxisomal targeting of a foreign enzyme puts some constraints to cellular viability. As a model system we used two different reporter constructs. In the first, expression of the luciferase gene is under control of CUP1-promoter, a well known yeast promoter that is inducible by copper ions. In the second, luciferase activity is dependent on activation of the human oestrogen receptor and its interaction with oestrogen-responsive elements incorporated in a yeast promoter. The luciferase activity measurement could be done on a 96-well plate by simple addition of the substrate, D-luciferin, at a moderately acidic pH of 5.0. The ease of use of the non-peroxisomal luciferase makes it an interesting alternative for reporter genes that are conventionally used in yeast, such as lacZ.     

Functional analysis of yeast essential genes using a promoter-substitution cassette and the tetracycline-regulatable dual expression system

A promoter-substitution cassette has been constructed that allows one-step substitution of chromosomal gene promoters for the tetracycline-regulatable tetO promoter in yeast cells, which uses kanMX4 as selective marker for geneticin resistance. Oligonucleotides for PCR amplification of the cassette are designed to allow homologous recombination through short flanking regions of homology with the upstream sequences of the chromosomal gene, upon transformation of target cells. By testing three essential genes of chromosome XV (YOL135c, YOL142w and YOL144w), the system causes tetracycline-dependent conditional growth of the cells, being modulatable by intermediate concentrations of the effector. Analysis of terminal phenotypes of the promoter-substituted cells in the presence of the antibiotic may facilitate functional analysis of essential orphan genes. © 1998 John Wiley & Sons, Ltd.     

An improved tetO promoter replacement system for regulating the expression of yeast genes

Regulatable promoters are commonly used to control the expression of, especially, essential genes in a conditional manner. Integration of such promoters upstream of an ORF using one-step PCR-mediated homologous recombination should be particularly efficient. However, integration of the original KanMX4-tetO promoter cassette (Belli et al., 1998a) into the relatively short upstream regions of many yeast genes is often problematic, presumably due to the size (3.9 kb) of the replacement cassette. We have created a new, shorter, KanMX4-tetO cassette by removing the transactivator (tTA) sequence from the original cassette. The transactivator (tTA) has been integrated into the yeast genome to create a new strain for use with the new system, which has a greatly increased efficiency of promoter substitution. With it, we have been able to create strains that could not be made with the original cassette. To increase the throughput of promoter substitutions, we have developed a new assay for testing doxycycline sensitivity, based on liquid culture using microtitre trays. Altogether, the components of this new 'tool kit' greatly increase the efficiency of systematic promoter substitutions.     

Construction of a cytosolic firefly luciferase reporter cassette for use in PCR‐mediated gene deletion and fusion in Saccharomyces cerevisiae

Monitoring promoter response to environmental changes using reporter systems has provided invaluable information regarding cellular state. With the development of in vivo luciferase reporter systems, inexpensive, sensitive and accurate promoter assays have been developed without the variability reported between in vitro samplings. Current luciferase reporter systems, however, are largely inflexible to modifications to the promoter of interest. To overcome problems in flexibility and stability of these expression vectors, we report the creation of a novel vector system which introduces a cytosol‐localized Photinus pyralis luciferase [LUC*(?SKL)] capable of one‐step, in vivo measurements into a promoter–reporter system via PCR‐based gene deletion and fusion. After introduction of the reporter under HUG1 promoter control, cytosolic localization was confirmed by fluorescence microscopy. The dose–response of this novel construct was then compared with that of a similar HUG1Δ::yEGFP1 promoter–reporter system and shown to give a similar response pattern. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitation of readthrough of termination codons in yeast using a novel gene fusion assay

A simple quantitative in vivo assay has been developed for measuring the efficiency of translation of one or other of the three termination codons. UAA, UAG and UGA in Saccharomyces cerevisiae. The assay employs a 3-phosphoglycerate kinase-beta-galactosidase gene fusion, carried on a multicopy plasmid, in which the otherwise retained reading frame is disrupted by one or other of the three termination codons. Termination readthrough is thus quantitated by measuring beta-galactosidase in transformed strains. Using these plasmids to quantitate the endogenous levels of termination readthrough we show that readthrough of all three codons can be detected in a non-suppressor (sup+) strain of S. cerevisiae. The efficiency of this endogenous readthrough is much higher in a [psi+] strain than in a [psi-] strain with the UGA codon being the leakiest in the nucleotide context used. The utility of the assay plasmids for studying genetic modifiers of nonsense suppressors is also shown by their use to demonstrate that the cytoplasmic genetic determinant [psi+] broadens the decoding properties of a serine-inserting UAA suppressor tRNA (SUQ5) to allow it to translate the other two termination codons in the order of efficiency UAA greater than UAG greater than UGA.     

Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has attracted much attention as a tool to study a number of biological processes. This study describes the use of GFP as a vital reporter molecule for localization and expression studies in Saccharomyces cerevisiae. Construction of GFP expression vectors which allow N- or C-terminal fusion of the gfp gene to a gene of interest allowed the generation of fusion proteins whose subcellular localization was followed by fluorescence microscopy in living yeast cells. Analysis of three unknown open reading frames obtained from the budding yeast chromosome XIV resulted in distinct staining patterns, allowing prediction of the cellular localization of these unknown proteins. Furthermore, GFP was used to construct a gene replacement cassette which, after homologous integration into the genomic locus, placed the gfp gene behind a promoter of interest. The amount of GFP produced from this promoter was then quantified in living yeast cells by flow cytometry. With this novel replacement cassette a gene of interest can be deleted and at the same time its expression level studied under various growth conditions. The experiments presented here suggest that GFP represents a convenient fluorescent marker for localization studies as well as gene expression studies in budding yeast. Systematic studies of a large number of genes should benefit from such assays.     

Increase in gene expression by respiratory-deficient mutation

Respiratory-deficient mutants (rho- cells) of Saccharomyces cerevisiae produced about 10 times as much human (h-) lysozyme as did wild-type strains (rho+ cells) when the GAL10 promoter was used in an expression plasmid with the h-lysozyme gene. Introduction of intact mitochondria into the rho- cells resulted in a significant decrease in the production of h-lysozyme, indicating that the rho- mutation increased the expression of the h-lysozyme gene. The copy number of the expression plasmid was not responsible for the increased expression. The level of h-lysozyme mRNA in the rho- cells was also much higher than that in the rho+ cells especially at the stationary phase. The increased expression of the h-lysozyme gene was also observed when a glyceraldehyde-3-phosphate dehydrogenase gene promoter and the PHO5 promoter were used in the expression plasmid. The rho- mutation also increased the expression of the PHO5 gene under the control of the HIS5 promoter in a plasmid and the ACT1 gene in the yeast chromosome, but did not increase the expression of the ribosomal RNA gene. In contrast to the rho- mutants, pet mutants did not show higher gene expression compared with wild-type strains.     

Regulation of the expression of endopolygalacturonase gene PGU1 in Saccharomyces

Previous work in our laboratory has shown that Saccharomyces bayanus strain SCPP is the only reported yeast expressing the three types of pectolytic enzymes: pectin esterases, pectin lyases and polygalacturonases. One of these enzymes, the endopolygalacturonase (endoPG), hydrolyses plant-specific polysaccharide pectin. The endoPG encoding gene (PGU1) is also present in Saccharomyces cerevisiae. It has been shown that this endoPG is required for the development of pseudohyphae. Using genomic DNA, the PGU1-1 and PGU1-2 promoters of these strains have been amplified and used to construct gene fusions with the beta-galactosidase gene. On the basis of beta-galactosidase measurements, we compared the expression of both promoters in different environmental conditions in order to identify their modulation. We have shown that the PGU1 gene is upregulated by the presence of the pectin and the product resulting from endopolygalacturonase activity. Moreover, expression of the PGU1 is also enhanced under respiratory and filament formation conditions.     

Cloning and characterization of a dextranase gene from Lipomyces starkeyi and its expression in Saccharomyces cerevisiae

A dextranase-encoding cDNA from L. starkeyi KSM22 was isolated and characterized. The 2052 bp cDNA fragment (lsd1) harbouring the dextranase gene exhibited one open reading frame (ORF) composed of 1824 bp flanked by a 41 bp 5'-UTR and a 184 bp 3'-UTR, including a 27 bp poly(A) tail. The lsd1 gene contains no introns. The open reading frame encodes a 608 amino acid polypeptide (LSD1) with a 67.6 kDa predicted molecular mass. There was a 77% deduced amino acid sequence identity between the LSD1 dextranase and the dextranase from Penicillium minioluteum. The primary structure of LSD1 dextranase exhibits distant similarity with the enzymes of the glycosyl hydrolase family 49 that comprises Penicillium dextranase. The optimum pH of LSD1 was 6.0 and the optimum temperature was 37 degrees C. LSD1 dextranase activity was substantially abolished by exposure to 1 mM Hg2+, Ag3+ and Mn2+. LSD1 exhibited high hydrolysing activity towards dextran (100%), soluble starch (22%) and mutan (8%).     

Identification of ACT4, a novel essential actin-related gene in the yeast Saccharomyces cerevisiae

Actin molecules are major cytoskeleton components of all eukaryotic cells. All conventional actins that have been identified so far are 374–376 amino acids in size and exhibit at least 70% amino acid sequence identity when compared with one another. In the yeast Saccharomyces cerevisiae, one conventional actin gene ACT1 and three so-called actin-related genes, ACT2, ACT3 and ACT5, have been identified. We report here the discovery of a new actin-related gene in this organism, which we have named ACT4. The deduced protein, Act4, of 449 amino acids, exhibits only 33·4%, 26·7%, 23·4% and 29·2% identity to Act1, Act2, Act3 and Act5, respectively. In contrast, it is 68·4% identical to the product of the Schizosaccharomyces pombe Act2 gene and has a similar level of identity to other Sch. pombe Act2 homologues. This places Act4 in the Arp3 family of actin-related proteins. ACT4 gene disruption and tetrad analysis demonstrate that this gene is essential for the vegetative growth of yeast cells. The act4 mutants exhibit heterogenous morphological phenotypes. We hypothesize that Act4 may have multiple roles in the cell cycle. The sequence has been deposited in the Genome Sequence Data Base under Accession Number L37111.     

A screening procedure for the intracellular expression of native proteins by Saccharomyces cerevisiae: discrimination of diphtheria toxin-resistant mutants.

A general method is described for screening Saccharomyces cerevisiae colonies for the intracellular expression of native proteins. Colonies are replicated onto nitrocellulose membranes and yeast cell walls are removed enzymatically. The resulting spheroplasts are rapidly lysed by placing chromatography paper soaked in hypotonic buffer on the membranes. Intracellular proteins released by spheroplast lysis are bound in situ to the nitrocellulose under non-denaturing conditions and potentially can be examined using enzymatic or immunologic methods. For example, in the present study colonies were screened for the presence of elongation factor 2 (EF-2) that can be [32P]ADP-ribosylated by diphtheria toxin and [32P]NAD+. Recognition by the toxin requires the presence in EF-2 of the unique post-translationally modified histidine derivative, diphthamide. The procedure described here reliably discriminates between wild-type yeast colonies and mutant colonies that do not synthesize diphthamide. In addition to facilitating the study of diphthamide biosynthesis in yeast, the more general application of this procedure will enable the screening of colonies with assays that require native proteins.     

The in vivo activation of Saccharomyces cerevisiae plasma membrane H+-ATPase by ethanol depends on the expression of the PMA1 gene,but not of the PMA2 gene

The expression of the PMA1 and PMA2 genes during Saccharomyces cerevisiae growth in medium with glucose plus increasing concentrations of ethanol was monitored by using PMA1-lacZ and PMA2-lacZ fusions and Northern blot hybridizations of total RNA probed with PMA1 gene. The presence of sub-lethal concentrations of ethanol enhanced the expression of PMA2 whereas it reduced the expression of PMA1. The inhibition of PMA1 expression by ethanol corresponded to a decrease in the content of plasma membrane ATPase as quantified by immunoassays. Although an apparent correspondence could exist between the increase of plasma membrane ATPase activity and the level of PMA2 expression, the maximal level of PMA2 expression remained about 200 times lower than PMA1. On the other hand, ethanol activated the plasma membrane H⁺-ATPase activity from a strain expressing only the PMA1 ATPase but did not activate that from a strain expressing only the PMA2 ATPase. These results provide evidence that in the presence of ethanol it is the PMA1 ATPase which is activated, probably by a post-translational mechanism and that the PMA2 ATPase is not involved.