scholarly journals Expression of Hygromycin Phosphotransferase Alters Virulence of Histoplasma capsulatum

2007 ◽  
Vol 6 (11) ◽  
pp. 2066-2071 ◽  
Author(s):  
A. George Smulian ◽  
Reta S. Gibbons ◽  
Jeffery A. Demland ◽  
Deborah T. Spaulding ◽  
George S. Deepe
Keyword(s):  
Mammalian Cells ◽  
Histoplasma Capsulatum ◽  
Fluorescent Protein ◽  
Selectable Marker ◽  
Animal Experiments ◽  
Yeast Cells ◽  
Growth And Survival ◽  
Hygromycin Phosphotransferase ◽  
Dominant Selectable Marker

ABSTRACT The Escherichia coli hygromycin phosphotransferase (hph) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the hph gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of Histoplasma capsulatum in the generation of knockout strains of H. capsulatum or as a marker in mutant strains. hph-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the hph gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of H. capsulatum. Infection of mice with hph-expressing H. capsulatum yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the hph construct into the genomes or to the method of integration and was not H. capsulatum strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the hph gene.

scholarly journals Cpc2, a Fission Yeast Homologue of Mammalian RACK1 Protein, Interacts with Ran1 (Pat1) Kinase To Regulate Cell Cycle Progression and Meiotic Development

2000 ◽  
Vol 20 (11) ◽  
pp. 4016-4027 ◽  
Author(s):  
Maureen McLeod ◽  
Boris Shor ◽  
Anthony Caporaso ◽  
Wei Wang ◽  
Hua Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Anchoring Protein ◽  
Signal Transduction Proteins ◽  
Green Fluorescent

ABSTRACT The Schizosaccharomyces pombe ran1/pat1 gene regulates the transition between mitosis and meiosis. Inactivation of Ran1 (Pat1) kinase is necessary and sufficient for cells to exit the cell cycle and undergo meiosis. The yeast two-hybrid interaction trap was used to identify protein partners for Ran1/Pat1. Here we report the identification of one of these, Cpc2. Cpc2 encodes a homologue of RACK1, a WD protein with homology to the β subunit of heterotrimeric G proteins. RACK1 is a highly conserved protein, although its function remains undefined. In mammalian cells, RACK1 physically associates with some signal transduction proteins, including Src and protein kinase C. Fission yeast cells containing a cpc2 null allele are viable but cell cycle delayed. cpc2Δ cells fail to accumulate in G1 when starved of nitrogen. This leads to defects in conjugation and meiosis. Copurification studies show that although Cpc2 and Ran1 (Pat1) physically associate, Cpc2 does not alter Ran1 (Pat1) kinase activity in vitro. Using a Ran1 (Pat1) fusion to green fluorescent protein, we show that localization of the kinase is impaired in cpc2Δ cells. Thus, in parallel with the proposed role of RACK1 in mammalian cells, fission yeast cpc2 may function as an anchoring protein for Ran1 (Pat1) kinase. All defects associated with loss of cpc2 are reversed in cells expressing mammalian RACK1, demonstrating that the fission yeast and mammalian gene products are indeed functional homologues.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

scholarly journals Efficient and Stable Delivery of Multiple Genes to Fish Cells by a Modified Recombinant Baculovirus System

2018 ◽  
Vol 19 (12) ◽  
pp. 3767 ◽  
Author(s):  
Qian Wang ◽  
Jian Fang ◽  
Qihua Pan ◽  
Yizhou Wang ◽  
Ting Xue ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Recombinant Baculovirus ◽  
Transgenic Fish ◽  
Early Gene ◽  
Drug Selection ◽  
Baculovirus System ◽  
Fish Cells

The recombinant baculovirus has been widely used as an efficient tool to mediate gene delivery into mammalian cells but has barely been used in fish cells. In the present study, we constructed a recombinant baculovirus containing the dual-promoter cytomegalovirus (CMV) and white spot syndrome virus (WSSV) immediate-early gene 1 (ie1) (WSSV ie1), followed by a puromycin–green fluorescent protein (Puro-GFP, pf) or puromycin–red fluorescent protein (Puro-RFP, pr) cassette, which simultaneously allowed for easy observation, rapid titer determination, drug selection, and exogenous gene expression. This recombinant baculovirus was successfully transduced into fish cells, including Mylopharyngodon piceus bladder (MPB), fin (MPF), and kidney (MPK); Oryzias latipes spermatogonia (SG3); and Danio rerio embryonic fibroblast (ZF4) cells. Stable transgenic cell lines were generated after drug selection, which was further verified by Western blot. A cell monoclonal formation assay proved the stable heredity of transgenic MPB cells. In addition, a recombinant baculovirus containing a pr cassette and four transcription factors for induced pluripotent stem cells (iPSC) was constructed and transduced into ZF4 cells, and these exogenous genes were simultaneously delivered and transcribed efficiently in drug-selected ZF4 cells, proving the practicability of this modified recombinant baculovirus system. We also proved that the WSSV ie1 promoter had robust activity in fish cells in vitro and in vivo. Taken together, this modified recombinant baculovirus can be a favorable transgenic tool to obtain transient or stable transgenic fish cells.

Supercharged green fluorescent protein delivers HPV16E7 DNA and protein into mammalian cells in vitro and in vivo

2018 ◽  
Vol 194 ◽  
pp. 29-39 ◽  
Author(s):  
Fatemeh Motevalli ◽  
Azam Bolhassani ◽  
Shilan Hesami ◽  
Sepideh Shahbazi
Keyword(s):  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals Characterization of hCINAP, a Novel Coilin-interacting Protein Encoded by a Transcript from the Transcription Factor TAFIID32 Locus

2005 ◽  
Vol 280 (43) ◽  
pp. 36429-36441 ◽  
Author(s):  
Niovi Santama ◽  
Stephen C. Ogg ◽  
Anna Malekkou ◽  
Spyros E. Zographos ◽  
Karsten Weis ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Essential Gene ◽  
Single Copy ◽  
Cajal Body ◽  
Cajal Bodies ◽  
Binding Motif

Coilin is a marker protein for the Cajal body, a subnuclear domain acting as a site for assembly and maturation of nuclear RNA-protein complexes. Using a yeast two-hybrid screen to identify coilin-interacting proteins, we have identified hCINAP (human coilin interacting nuclear ATPase protein), a nuclear factor of 172 amino acids with a P-loop nucleotide binding motif and ATPase activity. The hCINAP protein sequence is highly conserved across its full-length from human to plants and yeast and is ubiquitously expressed in all human tissues and cell lines tested. The yeast orthologue of CINAP is a single copy, essential gene. Tagged hCINAP is present in complexes containing coilin in mammalian cells and recombinant, Escherichia coli expressed hCINAP binds directly to coilin in vitro. The 214 carboxyl-terminal residues of coilin appear essential for the interaction with hCINAP. Both immunofluorescence and fluorescent protein tagging show that hCINAP is specifically nuclear and distributed in a widespread, diffuse nucleoplasmic pattern, excluding nucleoli, with some concentration also in Cajal bodies. Overexpression of hCINAP in HeLa cells results in a decrease in the average number of Cajal bodies per nucleus, consistent with it affecting either the stability of Cajal bodies and/or their rate of assembly. The hCINAP mRNA is an alternatively spliced transcript from the TAF9 locus, which encodes the basal transcription factor subunit TAFIID32. However, hCINAP and TAFIID32 mRNAs are translated from different ATG codons and use distinct reading frames, resulting in them having no identity in their respective protein sequences.

scholarly journals Cryptococcus neoformans Differential Gene Expression Detected In Vitro and In Vivo with Green Fluorescent Protein

1999 ◽  
Vol 67 (4) ◽  
pp. 1812-1820 ◽  
Author(s):  
Maurizio del Poeta ◽  
Dena L. Toffaletti ◽  
Thomas H. Rude ◽  
Sara D. Sparks ◽  
Joseph Heitman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Cns Infection ◽  
Gfp Expression ◽  
Gfp Reporter ◽  
Gfp Reporter Gene ◽  
Differential Gene ◽  
Green Fluorescent

ABSTRACT Synthetic green fluorescent protein (GFP) was used as a reporter to detect differential gene expression in the pathogenic fungusCryptococcus neoformans. Promoters from the C. neoformans actin, GAL7, or mating-type alpha pheromone (MFα1) genes were fused to GFP, and the resulting reporter genes were used to assess gene expression in serotype A C. neoformans. Yeast cells containing an integrated pACT::GFP construct demonstrated that the actin promoter was expressed during vegetative growth on yeast extract-peptone-dextrose medium. In contrast, yeast cells containing the inducible GAL7::GFP or MFα1::GFP reporter genes expressed significant GFP activity only during growth on galactose medium or V-8 agar, respectively. These findings demonstrated that the GAL7 and MFα1 promoters from a serotype D C. neoformans strain function when introduced into a serotype A strain. Because the MFα1 promoter is induced by nutrient deprivation and the MATα locus containing the MFα1 gene has been linked with virulence, yeast cells containing the pMFα1::GFP reporter gene were analyzed for GFP expression in the central nervous system (CNS) of immunosuppressed rabbits. In fact, significant GFP expression from the MFα1::GFP reporter gene was detected after the first week of a CNS infection. These findings suggest that there are temporal, host-specific cues that regulate gene expression during infection and that the MFα1 gene is induced during the proliferative stage of a CNS infection. In conclusion, GFP can be used as an effective and sensitive reporter to monitor specific C. neoformans gene expression in vitro, and GFP reporter constructs can be used as an approach to identify a novel gene(s) or to characterize known genes whose expression is regulated during infection.

scholarly journals Lecithin:Retinol Acyl Transferase (LRAT) induces the formation of lipid droplets

2019 ◽  
Author(s):  
Martijn R. Molenaar ◽  
Tsjerk A. Wassenaar ◽  
Kamlesh K. Yadav ◽  
Alexandre Toulmay ◽  
Muriel C. Mari ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Lipid Droplets ◽  
Neutral Lipids ◽  
Yeast Cells ◽  
Acyl Transferase ◽  
Hydrophobic Core ◽  
Retinyl Ester ◽  
Steryl Esters ◽  
Retinyl Esters

AbstractLipid droplets are unique and nearly ubiquitous organelles that store neutral lipids in a hydrophobic core, surrounded by a monolayer of phospholipids. The primary neutral lipids are triacylglycerols and steryl esters. It is not known whether other classes of neutral lipids can form lipid droplets by themselves. Here we show that production of retinyl esters by lecithin:retinol acyl transferase (LRAT) in yeast cells, incapable of producing triacylglycerols and steryl esters, causes the formation of lipid droplets. By electron microscopy, these lipid droplets are morphologically indistinguishable from those in wild-type cells. In silico and in vitro experiments confirmed the propensity of retinyl esters to segregate from membranes and to form lipid droplets. The hydrophobic N-terminus of LRAT displays preferential interactions with retinyl esters in membranes and promotes the formation of large retinyl ester-containing lipid droplets in mammalian cells. Our combined data indicate that the molecular design of LRAT is optimally suited to allow the formation of characteristic large lipid droplets in retinyl ester-storing cells.

scholarly journals Comparison of In Vitro Activities of Camptothecin and Nitidine Derivatives against Fungal and Cancer Cells

1999 ◽  
Vol 43 (12) ◽  
pp. 2862-2868 ◽  
Author(s):  
Maurizio Del Poeta ◽  
Shih-Fong Chen ◽  
Daniel Von Hoff ◽  
Christine C. Dykstra ◽  
Mansukh C. Wani ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Mammalian Cells ◽  
Topoisomerase I ◽  
Active Form ◽  
Antifungal Drugs ◽  
Water Soluble ◽  
Yeast Cells ◽  
Original Structure ◽  
Camptothecin Analogs

ABSTRACT The activities of a series of camptothecin and nitidine derivatives that might interact with topoisomerase I were compared against yeast and cancer cell lines. Our findings reveal that structural modifications to camptothecin derivatives have profound effects on the topoisomerase I-drug poison complex in cells. Although the water-soluble anticancer agents topotecan and irinotecan are less active than the original structure, camptothecin, other derivatives or analogs with substitutions that increase compound solubility have also increased antifungal activities. In fact, a water-soluble prodrug appears to penetrate into the cell and release its active form; the resulting effect in complex with Cryptococcus neoformanstopoisomerase I is a fungicidal response and also potent antitumor activity. Some of the compounds that are not toxic to wild-type yeast cells are extremely toxic to the yeast cells when the C. neoformans topoisomerase I target is overexpressed. With the known antifungal mechanism of a camptothecin-topoisomerase I complex as a cellular poison, these findings indicate that drug entry may be extremely important for antifungal activity. Nitidine chloride exhibits antifungal activity against yeast cells through a mechanism(s) other than topoisomerase I and appears to be less active than camptothecin analogs against tumor cells. Finally, some camptothecin analogs exhibit synergistic antifungal activity against yeast cells in combination with amphotericin B in vitro. Our results suggest that camptothecin and/or nitidine derivatives can exhibit potent antifungal activity and that the activities of camptothecin derivatives with existing antifungal drugs may be synergistic against pathogenic fungi. These new compounds, which exhibit potent antitumor activities, will likely require further structural changes to find more selective activity against fungal versus mammalian cells to hold promise as a new class of antifungal agents.

scholarly journals Selection against expression of the Escherichia coli gene gpt in hprt+ mouse teratocarcinoma and hybrid cells.

1987 ◽  
Vol 7 (11) ◽  
pp. 4139-4141 ◽  
Author(s):  
C Besnard ◽  
E Monthioux ◽  
J Jami
Keyword(s):  
Escherichia Coli ◽  
Positive Selection ◽  
Mammalian Cells ◽  
Selectable Marker ◽  
Hybrid Cells ◽  
Genetic Studies ◽  
Dominant Selectable Marker ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Mouse Teratocarcinoma

Thioxanthine is toxic for mammalian cells transformed by the dominant selectable marker gpt. It allowed us to select, in the presence of the endogenous hypoxanthine-guanine phosphoribosyltransferase gene, mutants that did not express gpt any more and also hybrid cells that had lost the chromosome carrying it. The gpt marker is thus dominant in negative as well as in positive selection, which makes it potentially very useful for genetic studies of mammalian cells.

scholarly journals Identification and Functional Characterization of a Novel Nuclear Localization Signal Present in the Yeast Nab2 Poly(A)+ RNA Binding Protein

1998 ◽  
Vol 18 (3) ◽  
pp. 1449-1458 ◽  
Author(s):  
Ray Truant ◽  
Robert A. Fridell ◽  
R. Edward Benson ◽  
Hal Bogerd ◽  
Bryan R. Cullen
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Yeast Cells ◽  
Vitro Assay ◽  
Localization Signal

ABSTRACT The nuclear import of proteins bearing a basic nuclear localization signal (NLS) is dependent on karyopherin α/importin α, which acts as the NLS receptor, and karyopherin β1/importin β, which binds karyopherin α and mediates the nuclear import of the resultant ternary complex. Recently, a second nuclear import pathway that allows the rapid reentry into the nucleus of proteins that participate in the nuclear export of mature mRNAs has been identified. In mammalian cells, a single NLS specific for this alternate pathway, the M9 NLS of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), has been described. The M9 NLS binds a transport factor related to karyopherin β1, termed karyopherin β2 or transportin, and does not require a karyopherin α-like adapter protein. A yeast homolog of karyopherin β2, termed Kap104p, has also been described and proposed to play a role in the nuclear import of a yeast hnRNP-like protein termed Nab2p. Here, we define a Nab2p sequence that binds to Kap104p and that functions as an NLS in both human and yeast cells despite lacking any evident similarity to basic or M9 NLSs. Using an in vitro nuclear import assay, we demonstrate that Kap104p can direct the import into isolated human cell nuclei of a substrate containing a wild-type, but not a defective mutant, Nab2p NLS. In contrast, other NLSs, including the M9 NLS, could not function as substrates for Kap104p. Surprisingly, this in vitro assay also revealed that human karyopherin β1, but not the Kap104p homolog karyopherin β2, could direct the efficient nuclear import of a Nab2p NLS substrate in vitro in the absence of karyopherin α. These data therefore identify a novel NLS sequence, active in both yeast and mammalian cells, that is functionally distinct from both basic and M9 NLS sequences.

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