Streptococcal Reporter Gene-Fusion Vector for Identification of in Vivo Expressed Genes

Plasmid ◽  
1999 ◽  
Vol 42 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Ali O. Kiliç ◽  
Mark C. Herzberg ◽  
Maurice W. Meyer ◽  
Xuemei Zhao ◽  
Lin Tao
Keyword(s):  
Reporter Gene ◽  
Gene Fusion ◽  
Reporter Gene Fusion

Environmentally regulated genes of Helicobacter pylori identified by a random lacZ reporter gene fusion system

1999 ◽  
Vol 11 (12) ◽  
pp. A98
Author(s):  
N. de Vries ◽  
E. J. Kuipers ◽  
A. H.M. van Vliet ◽  
N. E. Kramer ◽  
S. Bereswill ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Reporter Gene ◽  
Gene Fusion ◽  
Fusion System ◽  
Lacz Reporter ◽  
Lacz Reporter Gene ◽  
Reporter Gene Fusion

scholarly journals Isogenic Strain Construction and Gene Targeting inCandida dubliniensis

2001 ◽  
Vol 183 (9) ◽  
pp. 2859-2865 ◽  
Author(s):  
Peter Staib ◽  
Gary P. Moran ◽  
Derek J. Sullivan ◽  
David C. Coleman ◽  
Joachim Morschhäuser
Keyword(s):  
Reporter Gene ◽  
Gene Fusion ◽  
Recombinant Dna ◽  
Efflux Pump ◽  
Acid Resistance ◽  
Selection Marker ◽  
Targeted Gene Deletion ◽  
Yeast Extract Peptone Dextrose ◽  
Reporter Gene Fusion

ABSTRACT Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans but differs from it with respect to epidemiology, certain virulence characteristics, and the ability to develop fluconazole resistance in vitro. A comparison of C. albicans and C. dubliniensis at the molecular level should therefore provide clues about the mechanisms used by these two species to adapt to their human host. In contrast to C. albicans, no auxotrophic C. dubliniensis strains are available for genetic manipulations. Therefore, we constructed homozygous ura3 mutants from a C. dubliniensiswild-type isolate by targeted gene deletion. The two URA3alleles were sequentially inactivated using theMPAR -flipping strategy, which is based on the selection of integrative transformants carrying a mycophenolic acid resistance marker that is subsequently deleted again by site-specific, FLP-mediated recombination. The URA3 gene from C. albicans (CaURA3) was then used as a selection marker for targeted integration of a fusion between the C. dubliniensis MDR1 (CdMDR1) promoter and a C. albicans-adapted GFP reporter gene. Uridine-prototrophic transformants were obtained with high frequency, and all transformants of two independent ura3-negative parent strains had correctly integrated the reporter gene fusion into the CdMDR1 locus, demonstrating that the CaURA3gene can be used for efficient and specific targeting of recombinant DNA into the C. dubliniensis genome. Transformants carrying the reporter gene fusion did not exhibit detectable fluorescence during growth in yeast extract-peptone-dextrose medium in vitro, suggesting that CdMDR1 is not significantly expressed under these conditions. Fluconazole had no effect on MDR1 expression, but the addition of the drug benomyl strongly activated the reporter gene fusion in a dose-dependent fashion, demonstrating that theCdMDR1 gene, which encodes an efflux pump mediating resistance to toxic compounds, is induced by the presence of certain drugs.

scholarly journals Influence of a Functional sigB Operon on the Global Regulators sar and agr inStaphylococcus aureus

2001 ◽  
Vol 183 (17) ◽  
pp. 5171-5179 ◽  
Author(s):  
M. Bischoff ◽  
J. M. Entenza ◽  
P. Giachino
Keyword(s):  
Reporter Gene ◽  
Growth Phase ◽  
Gene Fusion ◽  
Firefly Luciferase ◽  
Luria Bertani ◽  
Transcriptional Analysis ◽  
Dependent Manner ◽  
Wild Type ◽  
Global Regulators ◽  
Reporter Gene Fusion

ABSTRACT The growth phase-dependent activity profile of the alternate transcription factor ςB and its effects on the expression of sar and agr were examined in three differentStaphylococcus aureus strains by Northern blot analyses and by the use of reporter gene fusion experiments. Significant ςB activity was detectable only in the clinical isolates MSSA1112 and Newman, carrying the wild-type rsbU allele, but not in the NCTC8325 derivative BB255, which is defective inrsbU. ςB activity peaked in the late exponential phase and diminished towards the stationary phase when bacteria were grown in Luria-Bertani medium. Transcriptional analysis and a sarP1-sarP2-sarP3(sarP1-P2-P3)-driven firefly luciferase (luc+) reporter gene fusion demonstrated a strong ςB activity- and growth phase-dependent increase in sar expression that was totally absent in either rsbU or ΔrsbUVWsigB mutants. In contrast, expression of theagr locus, as measured by RNAIII levels and by anhldp::luc+ fusion, was found to be higher in the absence of ςB activity, such as inrsbU or ΔrsbUVWsigB mutants, than in wild-type strains. Overexpression of ςB in BB255 derivatives resulted in a clear increase insarP1-P2-P3::luc+ expression as well as a strong decrease in hldp::luc+ expression. The data presented here suggest that ςBincreases sar expression while simultaneously reducing the RNAIII level in a growth phase-dependent manner.

Identification of Environmental Stress-Regulated Genes in Helicobacter pylori by a lacZ Reporter Gene Fusion System

Helicobacter ◽  
2001 ◽  
Vol 6 (4) ◽  
pp. 300-309 ◽  
Author(s):  
Nicolette de Vries ◽  
Ernst J. Kuipers ◽  
Naomi E. Kramer ◽  
Arnoud H. M. van Vliet ◽  
Jetta J. E. Bijlsma ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Environmental Stress ◽  
Reporter Gene ◽  
Gene Fusion ◽  
Fusion System ◽  
Lacz Reporter ◽  
Lacz Reporter Gene ◽  
Reporter Gene Fusion

scholarly journals Sir Proteins, Rif Proteins, and Cdc13p BindSaccharomyces Telomeres In Vivo

1998 ◽  
Vol 18 (9) ◽  
pp. 5600-5608 ◽  
Author(s):  
Brenda D. Bourns ◽  
Mary Kate Alexander ◽  
Andrew M. Smith ◽  
Virginia A. Zakian
Keyword(s):  
Reporter Gene ◽  
Transcriptional Activation ◽  
Binding Proteins ◽  
Position Effect ◽  
Initiation Site ◽  
Telomeric Sequence ◽  
Transcriptional Activation Domain ◽  
Amino Terminal ◽  
Sir Proteins

ABSTRACT Although a surprisingly large number of genes affect yeast telomeres, in most cases it is not known if their products act directly or indirectly. We describe a one-hybrid assay for telomere binding proteins and use it to establish that six proteins that affect telomere structure or function but which had not been shown previously to bind telomeres in vivo are indeed telomere binding proteins. A promoter-defective allele of HIS3 was placed adjacent to a chromosomal telomere. Candidate proteins fused to a transcriptional activation domain were tested for the ability to activate transcription of the telomere-linked HIS3 gene. Using this system, Rif1p, Rif2p, Sir2p, Sir3p, Sir4p, and Cdc13p were found to be in vivo telomere binding proteins. None of the proteins activated the same reporter gene when it was at an internal site on the chromosome. Moreover, Cdc13p did not activate the reporter gene when it was adjacent to an internal tract of telomeric sequence, indicating that Cdc13p binding was telomere limited in vivo. The amino-terminal 20% of Cdc13p was sufficient to target Cdc13p to a telomere, suggesting that its DNA binding domain was within this portion of the protein. Rap1p, Rif1p, Rif2p, Sir4p, and Cdc13p activated the telomeric reporter gene in a strain lacking Sir3p, which is essential for telomere position effect (TPE). Thus, the telomeric association of these proteins did not require any of the chromatin features necessary for TPE. The data support models in which the telomere acts as an initiation site for TPE by recruiting silencing proteins to the chromosome end.

In vitro and in vivo evaluation of a green fluorescent protein-HSV1-TK dual-function pet reporter gene

2001 ◽  
Vol 44 (S1) ◽  
pp. S339-S341
Author(s):  
K. E. Luker ◽  
G. D. Luker ◽  
C. M. Pica ◽  
J. L. Dahlheimer ◽  
T. J. Fahrner ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Reporter Gene ◽  
Fluorescent Protein ◽  
Dual Function ◽  
In Vivo Evaluation ◽  
Green Fluorescent

scholarly journals In vivo analysis of the myosin heavy chain IIB promoter region

1998 ◽  
Vol 274 (3) ◽  
pp. C681-C687 ◽  
Author(s):  
Steven J. Swoap
Keyword(s):  
Reporter Gene ◽  
Luciferase Activity ◽  
Fiber Type ◽  
Firefly Luciferase ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Base Pairs ◽  
Specific Expression ◽  
Mhc Iib

The myosin heavy chain (MHC) IIB gene is preferentially expressed in fast-twitch muscles of the hindlimb, such as the tibialis anterior (TA). The molecular mechanism(s) for this preferential expression are unknown. The goals of the current study were 1) to determine whether the cloned region of the MHC IIB promoter contains the necessary cis-acting element(s) to drive fiber-type-specific expression of this gene in vivo, 2) to determine which region within the promoter is responsible for fiber-type-specific expression, and 3) to determine whether transcription off of the cloned region of the MHC IIB promoter accurately mimics endogenous gene expression in a muscle undergoing a fiber-type transition. To accomplish these goals, a 2.6-kilobase fragment of the promoter-enhancer region of the MHC IIB gene was cloned upstream of the firefly luciferase reporter gene and coinjected with pRL-cytomegalovirus (CMV) (CMV promoter driving the renilla luciferase reporter) into the TA and the slow soleus muscle. Firefly luciferase activity relative to renilla luciferase activity within the TA was 35-fold greater than within the soleus. Deletional analysis demonstrated that only the proximal 295 base pairs (pGL3IIB0.3) were required to maintain this muscle-fiber-type specificity. Reporter gene expression of pGL3IIB0.3 construct was significantly upregulated twofold in unweighted soleus muscles compared with normal soleus muscles. Thus the region within the proximal 295 base pairs of the MHC IIB gene contains at least one element that can drive fiber-type-specific expression of a reporter gene.

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