scholarly journals Measles virus minigenomes encoding two autofluorescent proteins reveal cell-to-cell variation in reporter expression dependent on viral sequences between the transcription units

2007 ◽  
Vol 88 (10) ◽  
pp. 2710-2718 ◽  
Author(s):  
Linda J. Rennick ◽  
W. Paul Duprex ◽  
Bert K. Rima
Keyword(s):  
Gene Expression ◽  
Measles Virus ◽  
Fluorescent Protein ◽  
The Other ◽  
Green Fluorescent Protein Gene ◽  
Conserved Gene ◽  
Green Fluorescent ◽  
Single Living Cells ◽  
Cell Variation ◽  
Viral Sequences

Transcription from morbillivirus genomes commences at a single promoter in the 3′ non-coding terminus, with the six genes being transcribed sequentially. The 3′ and 5′ untranslated regions (UTRs) of the genes (mRNA sense), together with the intergenic trinucleotide spacer, comprise the non-coding sequences (NCS) of the virus and contain the conserved gene end and gene start signals, respectively. Bicistronic minigenomes containing transcription units (TUs) encoding autofluorescent reporter proteins separated by measles virus (MV) NCS were used to give a direct estimation of gene expression in single, living cells by assessing the relative amounts of each fluorescent protein in each cell. Initially, five minigenomes containing each of the MV NCS were generated. Assays were developed to determine the amount of each fluorescent protein in cells at both cell population and single-cell levels. This revealed significant variations in gene expression between cells expressing the same NCS-containing minigenome. The minigenome containing the M/F NCS produced significantly lower amounts of fluorescent protein from the second TU (TU2), compared with the other minigenomes. A minigenome with a truncated F 5′ UTR had increased expression from TU2. This UTR is 524 nt longer than the other MV 5′ UTRs. Insertions into the 5′ UTR of the enhanced green fluorescent protein gene in the minigenome containing the N/P NCS showed that specific sequences, rather than just the additional length of F 5′ UTR, govern this decreased expression from TU2.

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

2001 ◽  
Vol 67 (2) ◽  
pp. 948-955 ◽  
Author(s):  
Biao Ma ◽  
Mary B. Mayfield ◽  
Michael H. Gold
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Phanerochaete Chrysosporium ◽  
Fluorescent Protein ◽  
Schizophyllum Commune ◽  
Extracellular Fluid ◽  
Gene Promoter ◽  
Green Fluorescent Protein Gene ◽  
Cell Extracts ◽  
Green Fluorescent

ABSTRACT 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 egfpcoding region, and pUMGM3′ and pUMiGM3′ contain the P. chrysosporium mnp1 promoter fused upstream of theegfp 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 withegfp, 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 theegfp 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 theegfp 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 studyingcis-acting elements in the mnp1 gene promoter.

scholarly journals Effects of L1-ORF2 fragments on green fluorescent protein gene expression

2009 ◽  
Vol 32 (4) ◽  
pp. 688-696 ◽  
Author(s):  
Xiu-Fang Wang ◽  
Xia Jin ◽  
Xiaoyan Wang ◽  
Jing Liu ◽  
Jingjing Feng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Green Fluorescent

Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens

2006 ◽  
Vol 52 (7) ◽  
pp. 623-626 ◽  
Author(s):  
Weiguo Fang ◽  
Yan Pei ◽  
Michael J Bidochka
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Metarhizium Anisopliae ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Pathogenic Fungus ◽  
Single Copy ◽  
Transformation Method ◽  
The Other ◽  
Green Fluorescent Protein Gene ◽  
Green Fluorescent

A simple, highly efficient, and reliable Agrobacterium tumefaciens-mediated transformation method was developed for the insect pathogenic fungus Metarhizium anisopliae. Expression of the green fluorescent protein gene, egfp, and the benomyl resistance gene, benA3, were used as markers in transformed M. anisopliae. Transformation efficiencies were dependent on the strain of A. tumefaciens used. With strain AGL-1, 17.0 ± 1.4 transformants per plate could be obtained using conidial concentrations of 106 conidia/mL and a 2 day co-cultivation in the presence of 200 µmol/L acetosyringone. On the other hand, transformations using strain LBA4404 were unsuccessful. Ten transformants were tested by Southern analysis and found to contain a single copy T-DNA. Twenty transformants were subcultured for five generations on nonselective media, and 95% of the transformants were mitotically stable. Agrobacterium tumefaciens-mediated transformation of M. anisopliae can serve as a useful tool to investigate genes involved in insect pathogenicity.Key words: entomopathogenic fungi, Metarhizium anisopliae, Agrobacterium tumefaciens, genetic transformation.

scholarly journals Single-Copy Green Fluorescent Protein Gene Fusions Allow Accurate Measurement of Salmonella Gene Expression In Vitro and during Infection of Mammalian Cells

2003 ◽  
Vol 69 (12) ◽  
pp. 7480-7491 ◽  
Author(s):  
Isabelle Hautefort ◽  
Maria José Proença ◽  
Jay C. D. Hinton
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Single Copy ◽  
Green Fluorescent Protein Gene ◽  
Gene Fusions ◽  
Bacterial Gene ◽  
Green Fluorescent

ABSTRACT We developed a reliable and flexible green fluorescent protein (GFP)-based system for measuring gene expression in individual bacterial cells. Until now, most systems have relied upon plasmid-borne gfp gene fusions, risking problems associated with plasmid instability. We show that a recently developed GFP variant, GFP+, is suitable for assessing bacterial gene expression. Various gfp+ transcriptional fusions were constructed and integrated as single copies into the chromosome of Salmonella enterica serovar Typhimurium. A comparison of the expression levels of proU-lacZ and proU-gfp+ fusions showed that GFP+ reported proU activity in individual Salmonella cells as accurately as β-galactosidase reported activity for entire populations. The single-copy gfp+ fusions were ideal for monitoring up- and downregulation of Salmonella virulence genes. We discovered that in vitro induction of the SPI1gene prgH occurs only in a portion of the population and that the proportion varies with the growth phase. We determined the level of expression of the SPI2 gene ssaG in bacteria released from murine macrophages. Our results demonstrate for the first time that single-copy GFP+ fusions reliably report gene expression in simple and complex environments. This approach promises to allow accurate measurement of gene expression in individual bacteria during animal infection.

scholarly journals Essential and Dispensable Virus-Encoded Replication Elements Revealed by Efforts To Develop Hypoviruses as Gene Expression Vectors

2000 ◽  
Vol 74 (16) ◽  
pp. 7568-7577 ◽  
Author(s):  
Nobuhiro Suzuki ◽  
Lynn M. Geletka ◽  
Donald L. Nuss
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Cryphonectria Parasitica ◽  
Expression Vectors ◽  
Green Fluorescent Protein Gene ◽  
Reading Frame ◽  
Chestnut Blight Fungus ◽  
Virulence Attenuation ◽  
Foreign Genes ◽  
Green Fluorescent

ABSTRACT We have investigated whether hypoviruses, viral agents responsible for virulence attenuation (hypovirulence) of the chestnut blight fungusCryphonectria parasitica, could serve as gene expression vectors. The infectious cDNA clone of the prototypic hypovirus CHV1-EP713 was modified to generate 20 different vector candidates. Although transient expression was achieved for a subset of vectors that contained the green fluorescent protein gene from Aequorea victoria, long-term expression (past day 8) was not observed for any vector construct. Analysis of viral RNAs recovered from transfected fungal colonies revealed that the foreign genes were readily deleted from the replicating virus, although small portions of foreign sequences were retained by some vectors after months of replication. However, the results of vector viability and progeny characterization provided unexpected new insights into essential and dispensable elements of hypovirus replication. The N-terminal portion (codons 1 to 24) of the 5′-proximal open reading frame (ORF), ORF A, was found to be required for virus replication, while the remaining 598 codons of this ORF were completely dispensable. Substantial alterations were tolerated in the pentanucleotide UAAUG that contains the ORF A termination codon and the overlapping putative initiation codon of the second of the two hypovirus ORFs, ORF B. Replication competence was maintained following either a frameshift mutation that caused a two-codon extension of ORF A or a modification that produced a single-ORF genomic organization. These results are discussed in terms of determinants of hypovirus replication, the potential utility of hypoviruses as gene expression vectors, and possible mechanisms by which hypoviruses recognize and delete foreign sequences.

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