Detection of LacZ expression by FACS-Gal analysis

2008 ◽  
Author(s):  
Wei Guo ◽  
Hong Wu
Keyword(s):  
Lacz Expression

scholarly journals Transcriptional Regulators of theSchizosaccharomyces pombe fbp1Gene Include Two Redundant Tup1p-like Corepressors and the CCAAT Binding Factor Activation Complex

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1205-1215 ◽  
Author(s):  
Rozmin T K Janoo ◽  
Lori A Neely ◽  
Burkhard R Braun ◽  
Simon K Whitehall ◽  
Charles S Hoffman
Keyword(s):  
Protein Kinase ◽  
Fold Increase ◽  
Transcriptional Regulators ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Lacz Expression ◽  
Protein Subunit ◽  
Plasmid Library ◽  
Binding Factor ◽  
Mitogen Activated Protein

AbstractThe Schizosaccharomyces pombe fbp1 gene, which encodes fructose-1,6-bis-phosphatase, is transcriptionally repressed by glucose through the activation of the cAMP-dependent protein kinase A (PKA) and transcriptionally activated by glucose starvation through the activation of a mitogen-activated protein kinase (MAPK). To identify transcriptional regulators acting downstream from or in parallel to PKA, we screened an adh-driven cDNA plasmid library for genes that increase fbp1 transcription in a strain with elevated PKA activity. Two such clones express amino-terminally truncated forms of the S. pombe tup12 protein that resembles the Saccharomyces cerevisiae Tup1p global corepressor. These clones appear to act as dominant negative alleles. Deletion of both tup12 and the closely related tup11 gene causes a 100-fold increase in fbp1-lacZ expression, indicating that tup11 and tup12 are redundant negative regulators of fbp1 transcription. In strains lacking tup11 and tup12, the atf1-pcr1 transcriptional activator continues to play a central role in fbp1-lacZ expression; however, spc1 MAPK phosphorylation of atf1 is no longer essential for its activation. We discuss possible models for the role of tup11- and tup12-mediated repression with respect to signaling from the MAPK and PKA pathways. A third clone identified in our screen expresses the php5 protein subunit of the CCAAT-binding factor (CBF). Deletion of php5 reduces fbp1 expression under both repressed and derepressed conditions. The CBF appears to act in parallel to atf1-pcr1, although it is unclear whether or not CBF activity is regulated by PKA.

Two enhancer regions in the mouse En-2 locus direct expression to the mid/hindbrain region and mandibular myoblasts

Development ◽  
1993 ◽  
Vol 117 (3) ◽  
pp. 905-916 ◽  
Author(s):  
C. Logan ◽  
W.K. Khoo ◽  
D. Cado ◽  
A.L. Joyner
Keyword(s):  
Biochemical Analysis ◽  
Mutational Analysis ◽  
Expression Patterns ◽  
Genomic Region ◽  
Lacz Gene ◽  
Lacz Expression ◽  
Adult Mice ◽  
Granular Layers ◽  
Transcribed Sequences ◽  
Anterior Posterior

An En-2/lacZ gene fusion containing 9.5 kb of En-2 genomic DNA was capable of directing lacZ expression in an En-2-specific manner both temporally and spatially during embryogenesis and in the adult. lacZ expression was confined in the embryo to cells within the mid/hindbrain and mandibular arch regions and in the adult to cells of the molecular and granular layers of the cerebellum, and within the pons and colliculi regions. Interestingly, in the adult, transgene expression patterns within the cerebellum in two lines appeared to mark distinct anterior-posterior compartments. Analysis of the expression pattern of this transgene, in fetal and adult mice lacking a functional En-2 protein, provided evidence that the En-2 gene in mouse is not autoregulated. Deletion analysis of the En-2 genomic region and the use of a heterologous promoter identified two enhancer-containing regions of 1.5 and 1.0 kb in length, 5′ of the transcribed sequences, which independently directed expression in the embryo to either the mid/hindbrain region or mandibular myoblasts, respectively. The 1.5 kb fragment contains the most anterior neural enhancer and the 1.0 kb fragment, the earliest myogenic enhancer thus far characterized. These En-2-specific regulatory regions can now be used in a biochemical analysis to identify proteins important in anterior-posterior patterning of the vertebrate CNS and in the specification of muscle identity as well as in a mutational analysis to direct expression of other developmentally important genes to these regions.

scholarly journals Role of GCR2 in transcriptional activation of yeast glycolytic genes.

1992 ◽  
Vol 12 (9) ◽  
pp. 3834-3842 ◽  
Author(s):  
H Uemura ◽  
Y Jigami
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Gene Products ◽  
Lacz Expression ◽  
Genetic Method ◽  
Potential Interactions

The Saccharomyces cerevisiae GCR2 gene affects expression of most of the glycolytic genes. We report the nucleotide sequence of GCR2, which can potentially encode a 58,061-Da protein. There is a small cluster of asparagines near the center and a C-terminal region that would be highly charged but overall neutral. Fairly homologous regions were found between Gcr2 and Gcr1 proteins. To test potential interactions, the genetic method of S. Fields and O. Song (Nature [London] 340:245-246, 1989), which uses protein fusions of candidate gene products with, respectively, the N-terminal DNA-binding domain of Gal4 and the C-terminal activation domain II, assessing restoration of Gal4 function, was used. In a delta gal4 delta gal80 strain, double transformation by plasmids containing, respectively, a Gal4 (transcription-activating region)/Gcr1 fusion and a Gal4 (DNA-binding domain)/Gcr2 fusion activated lacZ expression from an integrated GAL1/lacZ fusion, indicating reconstitution of functional Gal4 through the interaction of Gcr1 and Gcr2 proteins. The Gal4 (transcription-activating region)/Gcr1 fusion protein alone complemented the defects of both gcr1 and gcr2 strains. Furthermore, a Rap1/Gcr2 fusion protein partially complemented the defects of gcr1 strains. These results suggest that Gcr2 has transcriptional activation activity and that the GCR1 and GCR2 gene products function together.

scholarly journals Inhibition of the multiple antibiotic resistance (mar) operon in Escherichia coli by antisense DNA analogs.

1997 ◽  
Vol 41 (12) ◽  
pp. 2699-2704 ◽  
Author(s):  
D G White ◽  
K Maneewannakul ◽  
E von Hofe ◽  
M Zillman ◽  
W Eisenberg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Antisense Oligonucleotides ◽  
Multiple Antibiotic Resistance ◽  
Wild Type ◽  
Lacz Expression ◽  
E Coli ◽  
Plasmid Construct ◽  
Dna Analogs ◽  
Susceptibility And Resistance

The multiple antibiotic resistance operon (marORAB) in Escherichia coli controls intrinsic susceptibility and resistance to multiple, structurally different antibiotics and other noxious agents. A plasmid construct with marA cloned in the antisense direction reduced LacZ expression from a constitutively expressed marA::lacZ translational fusion and inhibited the induced expression of LacZ in cells bearing the wild-type repressed fusion. The marA antisense construction also decreased the multiple antibiotic resistance of a Mar mutant. Two antisense phosphorothioate oligonucleotides, one targeted to marO and the other targeted to marA of the mar operon, introduced by heat shock or electroporation reduced LacZ expression in the strain having the marA::lacZ fusion. One antisense oligonucleotide, tested against a Mar mutant of E. coli ML308-225, increased the bactericidal activity of norfloxacin. These studies demonstrate the efficacy of exogenously delivered antisense oligonucleotides targeted to the marRAB operon in inhibiting expression of this chromosomal regulatory locus.

scholarly journals Localization of Glucagon-Like Peptide-2 Receptor Expression in the Mouse

Endocrinology ◽  
2019 ◽  
Vol 160 (8) ◽  
pp. 1950-1963 ◽  
Author(s):  
Bernardo Yusta ◽  
Dianne Matthews ◽  
Jacqueline A Koehler ◽  
Gemma Pujadas ◽  
Kiran Deep Kaur ◽  
...  
Keyword(s):  
Urinary Bladder ◽  
Lymph Nodes ◽  
Cellular Localization ◽  
Receptor Expression ◽  
Enteric Neurons ◽  
Lacz Expression ◽  
Mesenteric Lymph Nodes ◽  
Mesenteric Lymph ◽  
Mesenteric Fat ◽  
Glucagon Like Peptide

Abstract Glucagon-like peptide-2 (GLP-2), secreted from enteroendocrine cells, attenuates gut motility, enhances barrier function, and augments nutrient absorption, actions mediated by a single GLP-2 receptor (GLP-2R). Despite extensive analyses, the precise distribution and cellular localization of GLP-2R expression remains controversial, confounded by the lack of suitable GLP-2R antisera. Here, we reassessed murine Glp2r expression using regular and real-time quantitative PCR (qPCR), in situ hybridization (ISH), and a Glp2rLacZ reporter mouse. Glp2r mRNA expression was detected from the stomach to the rectum and most abundant in the jejunum. Glp2r transcripts were also detected in cerebral cortex, mesenteric lymph nodes, gallbladder, urinary bladder, and mesenteric fat. Surprisingly, Glp2r mRNA was found in testis by qPCR at levels similar to jejunum. However, the testis Glp2r transcripts, detected by different primer pairs and qPCR, lacked 5′ mRNA coding sequences, and only a minute proportion of them corresponded to full-length Glp2r mRNA. Within the gut, Glp2r-driven LacZ expression was localized to enteric neurons and lamina propria stromal cells, findings confirmed by ISH analysis of the endogenous Glp2r mRNA. Unexpectedly, vascular Glp2rLacZ expression was localized to mesenteric veins and not arteries. Moreover, mesenteric fat Glp2rLacZ expression was detected within blood vessels and not adipocytes. Reporter LacZ expression was not detected in all tissues expressing an endogenous Glp2r transcript, such as gallbladder, urinary bladder, and mesenteric lymph nodes. Collectively, these findings extend our understanding of the cellular domains of Glp2r expression and highlight limitations inherent in application of commonly used technologies to infer analysis of gene expression.

Inhibition of Wnt Pathway Signaling by Thalidomide and Revlimid: Studies in a Drosophila Model System.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3356-3356
Author(s):  
A. Keith Stewart ◽  
Yuan Xiao Zhu ◽  
Maryan Yahyapour ◽  
Armen Manoukian ◽  
Sam E. Scanga
Keyword(s):  
Wnt Signaling ◽  
Cell Lines ◽  
Wnt Pathway ◽  
High Throughput Sequencing ◽  
Cellular Proliferation ◽  
Wnt Signaling Pathway ◽  
Myeloma Cell ◽  
Lacz Expression ◽  
Western Blots ◽  
Drosophila Model

Abstract High throughput sequencing, gene expression profiling and protein biochemistry in myeloma have all consistently revealed elevated expression of wnt signaling pathways in malignant plasma cells. Indeed, downregulation of the Wnt pathway in myeloma cells has recently been shown to inhibit myeloma cellular proliferation. Preliminary pharmacogenomic studies have also suggested that hyperactivation of the wnt signaling antagonist DKK-1 is associated with response to the immunomodulators thalidomide and revlimid. The mechanism of action for these therapeutically active drugs is however by no means clear as multiple biologic consequences of treatment have been proposed. We report here use of a drosophila model to examine wnt signaling inhibition by these pharmaceuticals. We employed a unique drosophila larval imaginal disc culture system in which wnt pathway activity is monitored through control of LacZ expression by the distalless promoter. In this system 10uM of both thalidomide and revlimid reproducibly inhibit lacZ expression when compared with vehicle controls. Western blots of larva confirmed downregulation of expression of armadillo (the drosophila b-catenin homologue) by both drugs but particularly revlimid. Lithium Chloride is an inhibitor of the drosphila GSK3b homologue shaggy and thus mimics wnt signaling by stabilizing b-catenin. The effect of Lithium could not be overcome by thalidomide or revlimid indicating that the action of these drugs is upstream of shaggy (or GSK3). Next we employed a fly transgenic for wingless which is embryonic lethal. By adding either drug to larval culture medium the lethality of wingless expression was reversed. Indeed drosophila embryos fed thalidomide exhibited developmental plate abnormalities. We next sought evidence that similar effects were evident in revlimid treated human myeloma. As previously reported most myeloma cell lines studied expressed b-catenin and this protein was downregulated by revlimid treatment of human myeloma cell lines co-incident with inhibition of growth as measured by MTT assay. We sought, but failed to find evidence of up-regulation of the wnt signaling pathway antagonist DKK-1 using an ELISA assay on pre and post treatment serum samples in patients responding to thalidomide.The implications of wnt signaling inhibition as a primary or secondary readout of therapeutic efficiency in MM may be of substantial importance in subsequent design of drug therapies or combination therapies.

scholarly journals Scleraxis (Scx) directs lacZ expression in tendon of transgenic mice

2003 ◽  
Vol 120 (10) ◽  
pp. 1153-1163 ◽  
Author(s):  
Ana V Perez ◽  
Michael Perrine ◽  
Nicolas Brainard ◽  
Kathryn G Vogel
Keyword(s):  
Transgenic Mice ◽  
Lacz Expression
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