In Situ Histochemical Detection of β-Galactosidase Activity in Lung: Assessment of X-Gal Reagent in Distinguishing lacZ Gene Expression and Endogenous β-Galactosidase Activity

On exposure of Saccharomyces cerevisiae to sublethal concentrations of disinfectants such as TPN, Thiuram, Captan and Oxine-copper; 70-, 90-kDa proteins and heat-shock protein Hsp104 were induced without morphological changes. Considering these stress proteins as critical signs, we can determine how cells are damaged by pesticides under sublethal conditions. Furthermore, Hsp104-lacZ hybrid gene (a lacZ gene put under control of Hsp104 promoter) in S. cerevisiae was sensitively expressed in the presence of sublethal concentrations of these disinfectants by measuring the relative β-galactosidase activity. It follows that not only monitoring the growth phase or the induction of synthesized proteins but also detecting the level of gene expression shows the chemical stress response rapidly, conveniently and reproducibly. We conclude that the use of a yeast strain with a stress reporter gene is a novel and simple bioassay relative to human health and to the ecosystem in general.

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Homologous and heterologous enhancers modulate spatial expression but not cell-type specificity of the murine gamma F-crystallin promoter

Development ◽

10.1242/dev.110.1.131 ◽

1990 ◽

Vol 110 (1) ◽

pp. 131-139

Author(s):

C.C. Yu ◽

L.C. Tsui ◽

M.L. Breitman

Keyword(s):

Gene Expression ◽

Core Promoter ◽

Developmental Regulation ◽

Regulation Of Gene Expression ◽

Sufficient Information ◽

Lacz Gene ◽

Cell Type Specificity ◽

Fiber Cells

Previous studies have shown that mouse gamma F-crystallin sequences −759 to +45, which include the core promoter and two upstream enhancer elements, contain sufficient information for directing gene expression to terminally differentiated fiber cells of the ocular lens. To investigate the role that proximal sequences of the mouse gamma F-crystallin promoter play in the developmental regulation of gene expression, we generated transgenic mice containing the lacZ gene driven by either mouse gamma F-crystallin sequences −171 to +45, which lack functional enhancers, or a hybrid hamster alpha A-/mouse gamma F-crystallin promoter, which contains the hamster alpha A-crystallin enhancer instead of operational gamma F-crystallin enhancers. In situ analysis of lacZ expression in these mice revealed that the mouse gamma F-crystallin promoter segment −171 to +45, which shows low activity in vitro, is able to direct gene expression to the fiber cells in the nucleus of the lens. However, animals expressing gamma 171-lacZ show both a lower level of expression of the lacZ gene and a narrower pattern of staining in the lens nucleus than mice expressing gamma 759-lacZ, which contains the two enhancer elements located between −392 and −278 and −226 to −123.(ABSTRACT TRUNCATED AT 250 WORDS)

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In situ DNA transfer to chicken embryos by biolistics

Genetics and Molecular Biology ◽

10.1590/s1415-47571999000400010 ◽

1999 ◽

Vol 22 (4) ◽

pp. 525-529 ◽

Cited By ~ 1

Author(s):

Luciana A. Ribeiro ◽

João L. Azevedo ◽

Francisco J.L. Aragão ◽

Elibio L. Rech ◽

Gilberto S. Schmidt ◽

...

Keyword(s):

Survival Rate ◽

Dna Transfer ◽

Lacz Gene ◽

Galactosidase Activity ◽

Cmv Promoter ◽

Gold Particles ◽

Chicken Embryos ◽

Helium Gas ◽

Chicken Eggs

Fertilized chicken eggs were bombarded with a biolistic device. Transient expression of the lacZ gene under the control of a human cytomegalovirus (CMV) promoter was assessed after in situ gene transfer using this approach. The influence of different pressures, vacuum levels and particles was tested. Survival rate improved as particle velocity decreased, but resulted in lower levels of expression. The best survival and expression were obtained with gold particles, a helium gas pressure of 600 psi and a vacuum of 600 mmHg. Under these conditions, all bombarded embryos showed <FONT FACE="Symbol">b</FONT>-galactosidase activity, indicating that this was an effective method for transformation of chicken embryos.

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Functional organization of the Aspergillus nidulans trpC promoter

Molecular and Cellular Biology ◽

10.1128/mcb.7.7.2352-2359.1987 ◽

1987 ◽

Vol 7 (7) ◽

pp. 2352-2359

Author(s):

J E Hamer ◽

W E Timberlake

Keyword(s):

Gene Expression ◽

Aspergillus Nidulans ◽

Functional Organization ◽

Fusion Gene ◽

Lacz Gene ◽

Galactosidase Activity ◽

S1 Nuclease ◽

Promoter Mutations ◽

Beta Galactosidase ◽

Trpc Gene

We investigated the functional organization of the Aspergillus nidulans trpC promoter by the sequential removal of sequences upstream of the major trpC mRNA cap site (+1). DNA fragments containing promoter mutations were fused to the Escherichia coli lacZ gene, and a novel method was used to select for integration of the fusion gene at the Aspergillus argB locus. beta-Galactosidase assays and S1 nuclease protection experiments demonstrated that the promoter mutations affected gene expression in three ways: (i) 5' deletions up to -82 resulted in variable increases in beta-galactosidase activity, depending on the growth conditions; (ii) a deletion from -67 to -11 did not alter the level of beta-galactosidase activity, but did give rise to mRNAs with aberrant 5' ends; and (iii) a 5' deletion with an endpoint at -11 and an internal deletion from -142 to -11 abolished gene expression. These results indicate that sequences upstream of -82 reduce transcription of the trpC gene and that distinct DNA sequence elements are required for expression versus correct initiation of transcription of the trpC gene. The sequences essential for trpC expression do not include the common eucaryotic promoter elements CCAAT and TATAAA. To our knowledge, this is the first functional analysis of a promoter from a fungus other than Saccharomyces cerevisiae.

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In Situ Generated Novel 1H MRI Reporter for β-Galactosidase Activity Detection and Visualization in Living Tumor Cells

Frontiers in Chemistry ◽

10.3389/fchem.2021.709581 ◽

2021 ◽

Vol 9 ◽

Author(s):

Shuo Gao ◽

Lei Zhao ◽

Zhiqiang Fan ◽

Vikram D. Kodibagkar ◽

Li Liu ◽

...

Keyword(s):

Gene Expression ◽

Prostate Cancer ◽

Relaxation Times ◽

Small Animal ◽

Galactosidase Activity ◽

Activity Detection ◽

Prostate Cancer Cells ◽

Mri Contrast ◽

Clinical Assessments

For wide applications of the lacZ gene in cellular/molecular biology, small animal investigations, and clinical assessments, the improvement of noninvasive imaging approaches to precisely assay gene expression has garnered much attention. In this study, we investigate a novel molecular platform in which alizarin 2-O-β-d-galactopyranoside AZ-1 acts as a lacZ gene/β-gal responsive 1H-MRI probe to induce significant 1H-MRI contrast changes in relaxation times T1 and T2in situ as a concerted effect for the discovery of β-gal activity with the exposure of Fe3+. We also demonstrate the capability of this strategy for detecting β-gal activity with lacZ-transfected human MCF7 breast and PC3 prostate cancer cells by reaction-enhanced 1H-MRI T1 and T2 relaxation mapping.

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Functional organization of the Aspergillus nidulans trpC promoter.

Molecular and Cellular Biology ◽

10.1128/mcb.7.7.2352 ◽

1987 ◽

Vol 7 (7) ◽

pp. 2352-2359 ◽

Cited By ~ 76

Author(s):

J E Hamer ◽

W E Timberlake

Keyword(s):

Gene Expression ◽

Aspergillus Nidulans ◽

Functional Organization ◽

Fusion Gene ◽

Lacz Gene ◽

Galactosidase Activity ◽

S1 Nuclease ◽

Promoter Mutations ◽

Beta Galactosidase ◽

Trpc Gene

We investigated the functional organization of the Aspergillus nidulans trpC promoter by the sequential removal of sequences upstream of the major trpC mRNA cap site (+1). DNA fragments containing promoter mutations were fused to the Escherichia coli lacZ gene, and a novel method was used to select for integration of the fusion gene at the Aspergillus argB locus. beta-Galactosidase assays and S1 nuclease protection experiments demonstrated that the promoter mutations affected gene expression in three ways: (i) 5' deletions up to -82 resulted in variable increases in beta-galactosidase activity, depending on the growth conditions; (ii) a deletion from -67 to -11 did not alter the level of beta-galactosidase activity, but did give rise to mRNAs with aberrant 5' ends; and (iii) a 5' deletion with an endpoint at -11 and an internal deletion from -142 to -11 abolished gene expression. These results indicate that sequences upstream of -82 reduce transcription of the trpC gene and that distinct DNA sequence elements are required for expression versus correct initiation of transcription of the trpC gene. The sequences essential for trpC expression do not include the common eucaryotic promoter elements CCAAT and TATAAA. To our knowledge, this is the first functional analysis of a promoter from a fungus other than Saccharomyces cerevisiae.

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Molecular and Microscopic Analysis of Altered Gene Expression in Transgenic and Gene Targeted Mice

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162521 ◽

1996 ◽

Vol 54 ◽

pp. 12-13

Author(s):

W. K. Jones ◽

J. Robbins

Keyword(s):

Gene Expression ◽

Pulmonary Veins ◽

Microscopic Analysis ◽

Mouse Tissue ◽

Adult Heart ◽

Heavy Chains ◽

Altered Gene Expression ◽

Altered Gene ◽

Pulmonary Myocardium

Two myosin heavy chains (MyHC) are expressed in the mammalian heart and are differentially regulated during development. In the mouse, the α-MyHC is expressed constitutively in the atrium. At birth, the β-MyHC is downregulated and replaced by the α-MyHC, which is the sole cardiac MyHC isoform in the adult heart. We have employed transgenic and gene-targeting methodologies to study the regulation of cardiac MyHC gene expression and the functional and developmental consequences of altered α-MyHC expression in the mouse.We previously characterized an α-MyHC promoter capable of driving tissue-specific and developmentally correct expression of a CAT (chloramphenicol acetyltransferase) marker in the mouse. Tissue surveys detected a small amount of CAT activity in the lung (Fig. 1a). The results of in situ hybridization analyses indicated that the pattern of CAT transcript in the adult heart (Fig. 1b, top panel) is the same as that of α-MyHC (Fig. 1b, lower panel). The α-MyHC gene is expressed in a layer of cardiac muscle (pulmonary myocardium) associated with the pulmonary veins (Fig. 1c). These studies extend our understanding of α-MyHC expression and delimit a third cardiac compartment.

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RNA In Situ Hybridization for Detecting Gene Expression Patterns in the Abdomens and Wings of Drosophila Species

Methods and Protocols ◽

10.3390/mps4010020 ◽

2021 ◽

Vol 4 (1) ◽

pp. 20

Author(s):

Mujeeb Shittu ◽

Tessa Steenwinkel ◽

William Dion ◽

Nathan Ostlund ◽

Komal Raja ◽

...

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Expression Patterns ◽

Drosophila Species ◽

Gene Expression Patterns ◽

Probe Design ◽

Tissue Preparation ◽

Design And Synthesis ◽

Rna In Situ Hybridization

RNA in situ hybridization (ISH) is used to visualize spatio-temporal gene expression patterns with broad applications in biology and biomedicine. Here we provide a protocol for mRNA ISH in developing pupal wings and abdomens for model and non-model Drosophila species. We describe best practices in pupal staging, tissue preparation, probe design and synthesis, imaging of gene expression patterns, and image-editing techniques. This protocol has been successfully used to investigate the roles of genes underlying the evolution of novel color patterns in non-model Drosophila species.

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Growth, nisA Gene Expression, and In Situ Activity of Novel Lactococcus lactis subsp. cremoris Costarter Culture in Commercial Hard Cheese Production

Journal of Food Protection ◽

10.4315/0362-028x.jfp-17-245 ◽

2017 ◽

Vol 80 (12) ◽

pp. 2137-2146 ◽

Cited By ~ 7

Author(s):

Dimitrios Noutsopoulos ◽

Athanasia Kakouri ◽

Eleftheria Kartezini ◽

Dimitrios Pappas ◽

Efstathios Hatziloukas ◽

...

Keyword(s):

Gene Expression ◽

Lactococcus Lactis ◽

Starter Culture ◽

Fold Increase ◽

Raw Milk ◽

Good Growth ◽

Lactococcus Lactis Subsp ◽

Quantitative Expression ◽

Hard Cheese

ABSTRACT This study evaluated in situ expression of the nisA gene by an indigenous, nisin A–producing (NisA+) Lactococcus lactis subsp. cremoris raw milk genotype, represented by strain M78, in traditional Greek Graviera cheeses under real factory-scale manufacturing and ripening conditions. Cheeses were produced with added a mixed thermophilic and mesophilic commercial starter culture (CSC) or with the CSC plus strain M78 (CSC+M78). Cheeses were sampled after curd cooking (day 0), fermentation of the unsalted molds for 24 h (day 1), brining (day 7), and ripening of the brined molds (14 to 15 kg each) for 30 days in a fully controlled industrial room (16.5°C; 91% relative humidity; day 37). Total RNA was directly extracted from the cheese samples, and the expression of nisA gene was evaluated by real-time reverse transcription PCR (qRT-PCR). Agar overlay and well diffusion bioassays were correspondingly used for in situ detection of the M78 NisA+ colonies in the cheese agar plates and antilisterial activity in whole-cheese slurry samples, respectively. Agar overlay assays showed good growth (>8 log CFU/g of cheese) of the NisA+ strain M78 in coculture with the CSC and vice versa. The nisA expression was detected in CSC+M78 cheese samples only, with its expression levels being the highest (16-fold increase compared with those of the control gene) on day 1, followed by significant reduction on day 7 and almost negligible expression on day 37. Based on the results, certain intrinsic and mainly implicit hurdle factors appeared to reduce growth prevalence rates and decrease nisA gene expression, as well as the nisin A–mediated antilisterial activities of the NisA+ strain M78 postfermentation. To our knowledge, this is the first report on quantitative expression of the nisA gene in a Greek cooked hard cheese during commercial manufacturing and ripening conditions by using a novel, rarely isolated, indigenous NisA+ L. lactis subsp. cremoris genotype as costarter culture.

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