The green fluorescent protein gene functions as a reporter of gene expression in Phanerochaete chrysosporium

The enhanced green fluorescent protein (GFP) gene (egfp) was used as a reporter of gene expression driven by the glyceraldehyde-p-dehydrogenase (gpd) gene promoter and the manganese peroxidase isozyme 1 (mnp1) gene promoter in Phanerochaete chrysosporium. Four different constructs were prepared. pUGGM3' and pUGiGM3' contain the P. chrysosporium gpd promoter fused upstream of the egfp coding region, and pUMGM3' and pUMiGM3' contain the P. chrysosporium mnp1 promoter fused upstream of the egfp gene. In all constructs, the egfp gene was followed by the mnp1 gene 3' untranslated region. In pUGGM3' and pUMGM3', the promoters were fused directly with egfp, whereas in pUGiGM3' and pUMiGM3', following the promoters, the first exon (6 bp), the first intron (55 bp), and part of the second exon (9 bp) of the gpd gene were inserted at the 5' end of the egfp gene. All constructs were ligated into a plasmid containing the ura1 gene of Schizophyllum commune as a selectable marker and were used to transform a Ural1 auxotrophic strain of P. chrysosporium to prototrophy. Crude cell extracts were examined for GFP fluorescence, and where appropriate, the extracellular fluid was examined for MnP activity. The transformants containing a construct with an intron 5' of the egfp gene (pUGiGM3' and pUMiGM3') exhibited maximal fluorescence under the appropriate conditions. The transformants containing constructs with no introns exhibited minimal or no fluorescence. Northern (RNA) blots indicated that the insertion of a 5' intron resulted in more egfp RNA than was found in transformants carrying an intronless egfp. These results suggest that the presence of a 5' intron affects the expression of the egfp gene in P. chrysosporium. The expression of GFP in the transformants carrying pUMiGM3' paralled the expression of endogenous mnp with respect to nitrogen and Mn levels, suggesting that this construct will be useful in studying cis-acting elements in the mnp1 gene promoter.     

Green fluorescent protein as a marker for monitoring activity of stress-inducible hsp70 rat gene promoter

Murine melanoma cells B16(F10) were stably transfected with a plasmid containing GFP gene linked to rat stress-inducible hsp70.1 gene promoter. Transfected cells show in vitro variable basal levels of fluorescence depending on stress response induced at physiological temperature by growth conditions. Lack of manipulations except medium change resulted in reduction of cellular fluorescence. GFP expression in experimental murine tumors dropped to levels undetectable at physiological temperature. Heat shock induced significant fluorescence of tumor cells both in vitro and in vivo. GFP protein could be a useful marker for studies of mammalian hsp70i gene promoters.     

Novobiocin inhibits the SV40 enhancer activity

Using a transient expression assay, we have analysed the effect of novobiocin, DNA topoisomerase II inhibitor, on simian virus 40(SV40) enhancer activities. We used the recombinant clones containing type I or II collagen promoters placed upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene with or without SV40 enhancer. We observed the expected increase in CAT activities due to the presence of the SV40 enhancer. Interestingly, CAT gene expression of the enhancer-containing constructs were inhibited more sensitively by novobiocin than that of the enhancer-less construct. This findings lead us propose that DNA superhelicity mediated by topoisomeraseII is one of the important factor for the manifestation of SV40 enhancer activity.     

The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein

Mycobacterium marinum , like Mycobacterium tuberculosis , is a slow-growing pathogenic mycobacteria that is able to survive and replicate in macrophages. Using the promoter-capture vector pFPV27, we have constructed a library of 200–1000 bp fragments of M. marinum genomic DNA inserted upstream of a promoterless green fluorescent protein (GFP) gene. Only those plasmids that contain an active promoter will express GFP. Macrophages were infected with this fusion library, and phagosomes containing fluorescent bacteria were isolated. Promoter constructs that were more active intracellularly were isolated with a fluorescence-activated cell sorter, and inserts were partially sequenced. The promoter fusions expressed intracellularly exhibited homology to mycobacterial genes encoding, among others, membrane proteins and biosynthetic enzymes. Intracellular expression of GFP was 2–20 times that of the same clones grown in media. Several promoter constructs were transformed into Mycobacterium smegmatis , Mycobacterium bovis BCG and Mycobacterium tuberculosis . These constructs were positive for GFP expression in all mycobacterial strains tested. Sorting fluorescent bacteria in phagosomes circumvents the problem of isolating a single clone from macrophages, which may contain a mixed bacterial population. This method has enabled us to isolate 12 M. marinum clones that contain promoter constructs differentially expressed in the macrophage.     

Characterization of Promoter Activities of Four Different Japanese Flounder Promoters in Transgenic Zebrafish

An important consideration in transgenic research is the choice of promoter for regulating the expression of a foreign gene. In this study several tissue-specific and inducible promoters derived from Japanese flounder Paralichthys olivaceus were identified, and their promoter activity was examined in transgenic zebrafish. The 5′ flanking regions of the Japanese flounder complement component C3, gelatinase B, keratin, and tumor necrosis factor (TNF) genes were linked to green fluorescence protein (GFP) as a reporter gene. The promoter regulatory constructs were introduced into fertilized zebrafish eggs. As a result we obtained several stable transgenic zebrafish that displayed green fluorescence in different tissues. Complement component C3 promoter regulated GFP expression in liver, and gelatinase B promoter regulated it in the pectoral fin and gills. Keratin promoter regulated GFP expression in skin and liver. TNF gene promoter regulated GFP expression in the pharynx and heart. TNF promoter had lipoplysaccharide-inducible activity, such that when transgenic embryos were immersed lipopolysaccharide, GFP expression increased in the epithelial tissues. These 4 promoters regulated the expression of GFP in different patterns in transgenic zebrafish.     

Cnidarian and bilaterian promoters can direct GFP expression in transfected hydra

Complete sexual development is not easily amenable to experimentation in hydra. Therefore, the analysis of gene function and gene regulation requires the introduction of exogenous DNA in a large number of cells of the hydra polyps and the significant expression of reporter constructs in these cells. We present here the procedure whereby we coupled DNA injection into the gastric cavity to electroporation of the whole animal in order to efficiently transfect hydra polyps. We could detect GFP fluorescence in both endodermal and ectodermal cell layers of live animals and in epithelial as well as interstitial cell types of dissociated hydra. In addition, we could confirm GFP protein expression by showing colocalisation between GFP fluorescence and anti-GFP immunofluorescence. Finally, when a FLAG epitope was inserted in-frame with the GFP coding sequence, GFP fluorescence also colocalised with anti-FLAG immunofluorescence. This GFP expression in hydra cells was directed by various promoters, either homologous, like the hydra homeobox cnox-2 gene promoter, or heterologous, like the two nematode ribosomal protein S5 and L28 gene promoters, and the chicken beta-actin gene promoter. This strategy provides new tools for dissecting developmental molecular mechanisms in hydra; more specifically, the genetic regulations that take place in endodermal cells at the time budding or regeneration is initiated.     

Strongyloides stercoralis: cell- and tissue-specific transgene expression and co-transformation with vector constructs incorporating a common multifunctional 3' UTR

Transgenesis is a valuable methodology for studying gene expression patterns and gene function. It has recently become available for research on some parasitic nematodes, including Strongyloides stercoralis. Previously, we described a vector construct, comprising the promoter and 3' UTR of the S. stercoralis gene Ss era-1 that gives expression of GFP in intestinal cells of developing F1 progeny. In the present study, we identified three new S. stercoralis promoters, which, in combination with the Ss era-1 3' UTR, can drive expression of GFP or the red fluorescent protein, mRFPmars, in tissue-specific fashion. These include Ss act-2, which drives expression in body wall muscle cells, Ss gpa-3, which drives expression in amphidial and phasmidial neurons and Ss rps-21, which drives ubiquitous expression in F1 transformants and in the gonads of microinjected P0 female worms. Concomitant microinjection of vectors containing GFP and mRFPmars gave dually transformed F1 progeny, suggesting that these constructs could be used as co-injection markers for other transgenes of interest. We have developed a vector "toolkit" for S. stercoralis including constructs with the Ss era-1 3' UTR and each of the promoters described above.     

The Green Fluorescent Protein Gene Functions as a Reporter of Gene Expression in Phanerochaete chrysosporium

The enhanced green fluorescent protein (GFP) gene (egfp) was used as a reporter of gene expression driven by the glyceraldehyde-p-dehydrogenase (gpd) gene promoter and the manganese peroxidase isozyme 1 (mnp1) gene promoter in Phanerochaete chrysosporium. Four different constructs were prepared. pUGGM3′ and pUGiGM3′ contain the P. chrysosporium gpd promoter fused upstream of the egfp coding region, and pUMGM3′ and pUMiGM3′ contain the P. chrysosporium mnp1 promoter fused upstream of the egfp gene. In all constructs, the egfp gene was followed by the mnp1 gene 3′ untranslated region. In pUGGM3′ and pUMGM3′, the promoters were fused directly with egfp, whereas in pUGiGM3′ and pUMiGM3′, following the promoters, the first exon (6 bp), the first intron (55 bp), and part of the second exon (9 bp) of the gpd gene were inserted at the 5′ end of the egfp gene. All constructs were ligated into a plasmid containing the ura1 gene of Schizophyllum commune as a selectable marker and were used to transform a Ural1 auxotrophic strain of P. chrysosporium to prototrophy. Crude cell extracts were examined for GFP fluorescence, and where appropriate, the extracellular fluid was examined for MnP activity. The transformants containing a construct with an intron 5′ of the egfp gene (pUGiGM3′ and pUMiGM3′) exhibited maximal fluorescence under the appropriate conditions. The transformants containing constructs with no introns exhibited minimal or no fluorescence. Northern (RNA) blots indicated that the insertion of a 5′ intron resulted in more egfp RNA than was found in transformants carrying an intronless egfp. These results suggest that the presence of a 5′ intron affects the expression of the egfp gene in P. chrysosporium. The expression of GFP in the transformants carrying pUMiGM3′ paralled the expression of endogenous mnp with respect to nitrogen and Mn levels, suggesting that this construct will be useful in studying cis-acting elements in the mnp1 gene promoter.     

Identification and characterization of novel and potent transcription promoters of Francisella tularensis

Two alternative promoter trap libraries, based on the green fluorescence protein (gfp) reporter and on the chloramphenicol acetyltransferase (cat) cassette, were constructed for isolation of potent Francisella tularensis promoters. Of the 26,000 F. tularensis strain LVS gfp library clones, only 3 exhibited visible fluorescence following UV illumination and all appeared to carry the bacterioferritin promoter (Pbfr). Out of a total of 2,000 chloramphenicol-resistant LVS clones isolated from the cat promoter library, we arbitrarily selected 40 for further analysis. Over 80% of these clones carry unique F. tularensis DNA sequences which appear to drive a wide range of protein expression, as determined by specific chloramphenicol acetyltransferase (CAT) Western dot blot and enzymatic assays. The DNA sequence information for the 33 unique and novel F. tularensis promoters reported here, along with the results of in silico and primer extension analyses, suggest that F. tularensis possesses classical Escherichia coli σ(70)-related promoter motifs. These motifs include the -10 (TATAAT) and -35 [TTGA(C/T)A] domains and an AT-rich region upstream from -35, reminiscent of but distinct from the E. coli upstream region that is termed the UP element. The most efficient promoter identified (Pbfr) appears to be about 10 times more potent than the F. tularensis groEL promoter and is probably among the strongest promoters in F. tularensis. The battery of promoters identified in this work will be useful, among other things, for genetic manipulation in the background of F. tularensis intended to gain better understanding of the mechanisms involved in pathogenesis and virulence, as well as for vaccine development studies.     

Libraries of synthetic stationary-phase and stress promoters as a tool for fine-tuning of expression of recombinant proteins in Escherichia coli

Due to their induction characteristics stationary-phase promoters have a great potential in biotechnological processes for the production of heterologous proteins on a large-scale. In order to broaden the utility of stationary-phase promoters in bacterial expression systems and to create novel promoters induced by metabolic conditions, a library of synthetic stationary-phase/stress promoters for Escherichia coli was constructed. For designing the promoters the known -10 consensus sequence as well as the extended -10 region and an A/T-rich region downstream of the -10 region were kept constant, while sequences from -37 to -14 were partially or completely randomized. For detection and selection of stationary-phase promoters GFP with enhanced fluorescence was used. The expression pattern of the GFP reporter system was compared with that of the LacZ reporter system. To screen and characterize colonies containing stationary-phase/stress promoters a bioinformatic approach was developed. In total, 33 promoters were selected which cover a broad range of promoter activities and induction times indicating that the strength of promoters can be modulated by partially randomizing the sequence upstream of the -10 region. The induction ratio of synthetic promoters at the transition from exponential to stationary-phase was from 4 to over 6000 and the induction time relative to the entrance into stationary-phase from -1.4 to 2.7 h. Ninety-one percentage of the promoters had no or only low background activity during exponential growth. The broad variability of the promoters offers good possibilities for fine-tuning of gene expression and for applications in industrial bioprocesses.     

Firefly luciferase gene transmission and expression in transgenic medaka (Oryzias latipes).

Plasmids containing the luciferase gene from the firefly (Photinus pyralis) fused to the Chinese hamster metallothioneine I promoter (ChMTI) were microinjected into the pronuclei of medaka (Oryzias latipes) eggs, which were then artificially inseminated. Evidence of integration into the genome was gained from observation of germ-line transmission in a mendelian fashion from the F1 to the F2 generation. However, gene expression (light emission) could not be demonstrated in the established transgenic line. In a separate program, transient expression of gene constructs containing the luciferase gene fused to various promoters was compared in medaka embryos. Plasmids were microinjected into pronuclei, and homogenates from 3-day-old embryos were measured for light emission using a luminometer. Among the various promoters tested (SV40, RSV-LTR, ChMTI, HSP70, and mouse albumin), the highest levels of luciferase gene expression were observed in gene constructs containing ChMTI and HSP70 gene promoters. Expression in these two constructs was significantly increased following administration of ZnSO4 or heat treatment, respectively. Plasmids were also introduced into goldfish fibroblast-like cells in vitro, in which enzymatically active luciferase was transiently expressed. Assaying for expression of luciferase provided a rapid and sensitive method for monitoring promoter activity. The potential usefulness of this fish species for cancer research is discussed based on accumulated information from carcinogenesis studies.     

Selection of transgenic Xenopus laevis using antibiotic resistance

We previously established lines of transgenic Xenopus laevis expressing green fluorescent protein (GFP) or GFP fusion proteins in the rod photoreceptors of their retinas under control of the X. laevis opsin promoter, which permits easy identification of transgenic animals by fluorescence microscopy. However, GFP tags can alter the properties of fusion partners, and in many circumstances a second selectable marker would be useful. The transgene constructs we used also encode a gene that confers resistance to the antibiotic G418 in cultured mammalian cells. In this study, we show that F2 transgenic offspring of these animals are more resistant to G418 toxicity than their non-transgenic siblings, as are primary transgenic X. laevis. G418 resistance can be used as a selectable marker in transgenic X. laevis, and possibly other aquatic transgenic animals.