Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phage Mu's c gene product is a cooperative regulatory protein that binds to a large, complex, tripartite 184-bp operator. To probe the mechanism of repressor action, we isolated and characterized 13 phage mutants that cause Mu to undergo lytic development when cells are shifted from 30 to 42 degrees C. This collection contained only four mutations in the repressor gene, and all were clustered near the N terminus. The cts62 substitution of R47----Q caused weakened specific DNA recognition and altered cooperativity in vitro. A functional repressor with only 63 amino acids of Mu repressor fused to a C-terminal fragment of beta-galactosidase was constructed. This chimeric protein was an efficient repressor, as it bound specifically to Mu operator DNA in vitro and its expression conferred Mu immunity in vivo. A DNA looping model is proposed to explain regulation of the tripartite operator site and the highly cooperative nature of repressor binding.
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PMID:Temperature-sensitive mutations in the bacteriophage Mu c repressor locate a 63-amino-acid DNA-binding domain. 183 82

Cascade polymers also known as Starburst dendrimers are spheroidal polycations that can be synthesized with a well-defined diameter and a precise number of terminal amines per dendrimer. We show, using luciferase and beta-galactosidase containing plasmids, that dendrimers mediate high efficiency transfection of a variety of suspension and adherent cultured mammalian cells. Dendrimer-mediated transfection is a function both of the dendrimer/DNA ratio and the diameter of the dendrimer. Maximal transfection of luciferase are obtained using a diameter of 68 A and a dendrimer to DNA charge ratio of 6/1 (terminal amine to phosphate). Expression is unaffected by lysomotrophic agents such as chloroquine and only modestly affected (2-fold decrease) by the presence of 10% serum in the medium. Cell viability, as assessed by dye reduction assays, decreases by only 30% at 150 micrograms dendrimer/mL in the absence of DNA and about 75% in the presence of DNA. Under similar conditions polylysine causes a complete loss of viability. Gene expression decreased by 3 orders of magnitude when the charge ratio is reduced to 1:1. When GALA, a water soluble, membrane-destabilizing peptide, is covalently attached to the dendrimer via a disulfide linkage, transfection efficiency of the 1:1 complex is increased by 2-3 orders of magnitude. The high transfection efficiency of the dendrimers may not only be due to their diameter and shape but may also be caused by the pKa's (3.9 and 6.9) of the amines in the polymer. The low pKa's permit the dendrimer to buffer the pH change in the endosomal compartment. The characteristics of precise control of structure, favorable pKa's, and low toxicity make the dendrimers suitable for gene-transfer vehicles.
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PMID:Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. 827 23

Starting from strain of Salmonella typhimurium purD::lac, 86 exponential cultures were mutagenized with NTG and white or light blue clones on E + ado + Xgal plate were selected as candidates of purRs mutant. Total 66 independent candidate strains were obtained. By assaying their beta-galactosidase activity under the repressed and derepressed conditions, determining their frequency of revertional mutation, Conducting transductional analysis of mutational site and dorminance test, 11 candidates strains were proved to be super-repressor mutants. These mutants are useful for studying the expression of purine biosynthetic gene and relationship between protein structure and function in general.
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PMID:[Expression regulation of purine biosynthetic genes in Salmonella typhimurium. VI. Isolation and characterization of super-repressor mutants]. 975 10

A versatile system consisting of an integrational vector and a bacitracin (Bt)-producing beta-galactosidase (beta-Gal)-negative (Lac-) Bacillus licheniformis TLH strain was constructed to quantify promoter activity and to study gene regulation in a single-copy set-up. The vector pTLH utilizes the promoterless Escherichia coli lacZ gene derived from pQF52 and contains the pBR322 origin of replication and a kanamycin-resistance gene for selection in both B. licheniformis and E. coli. The vector also contains an inner part of the first gene of the Bt synthetase (bts) operon which enables its integration into the bts of B. licheniformis by Campbell-type recombination. This recombination event can be easily tested on a Micrococcus flavus lawn where loss of Bt production, i.e. no clearing zone on the lawn, is indicative of the proper integration. The Lac- B. licheniformis TLH strain was developed by elimination of the natural beta-Gal activity of B. licheniformis strain ATCC 10716 UM12 using NTG mutagenesis.
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PMID:Construction of a single-copy integration vector and its use to study gene expression in Bacillus licheniformis. 978 6

The serine/threonine protein kinase Akt (protein kinase B) phosphorylates endothelial cell nitric oxide synthase (eNOS) and enhances its ability to generate nitric oxide (NO). Because NO is an important regulator of vasomotor tone, we investigated whether Akt can regulate endothelium-dependent vasomotion in vivo using a rabbit femoral artery model of gene transfer. The endothelium of isolated femoral arteries was infected with replication-defective adenoviral constructs expressing beta-galactosidase, constitutively-active Akt (myr-Akt), or dominant-negative Akt (dn-Akt). Femoral arteries transduced with myr-Akt showed a significant increase in resting diameter and blood flow, as assessed by angiography and Doppler flow measurements, respectively. L-NAME, an eNOS inhibitor, blocked myr-Akt-mediated vasodilatation. In contrast, endothelium-dependent vasodilatation in response to acetylcholine was attenuated in vessels transduced with dn-Akt, although these vessels showed normal responses to nitroglycerin, an endothelium-independent vasodilator. Similarly, relaxation of murine aorta ex vivo in response to acetylcholine, but not nitroglycerin, was inhibited by transduction of dn-Akt to the endothelium. These data provide evidence that Akt functions as key regulator of vasomotor tone in vivo.
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PMID:Acute modulation of endothelial Akt/PKB activity alters nitric oxide-dependent vasomotor activity in vivo. 1095 24

Because of the lack of pharmacological approaches, molecular genetic methods have been required to differentiate between angiotensin type 1(AT1) receptor subtypes AT1a and AT1b. RNA interference is a new tool for the study of gene function, producing specific downregulation of protein expression. In this study, we used the small hairpin RNA (shRNA) cassette method to screen target sites for selectively silencing AT1a or AT1b receptor subtypes in cultured Neuro-2a cells using real-time RT-PCR. For in vivo functional studies, we used C57BL mice with arterial telemetric probes and computerized licking monitors to test the effect of adenovirus carrying the DNA sequence coding AT1a shRNA (Ad-AT1a-shRNA). Ad-AT1a-shRNA was injected into the lateral ventricle (intracerebroventricular) or the brain stem nucleus tractus solitaries/dorsal vagal nucleus (NTS/DVN) with measurement of water intake, blood pressure (BP), and heart rate (HR) for up to 20 days after injection. Tissue culture studies verified the specificity and the efficiency of the constructs. In animal studies, beta-galactosidase staining and Ang receptor binding assays showed expression of shRNA and downregulation of Ang AT1 receptors in the subfornical organ and NTS/DVN by >70%. Intracerebroventricular injection of Ad-AT1a-shRNA increased water intake with no effect on BP or HR. In contrast, microinjection of Ad-AT1a-shRNA into NTS/DVN caused a decrease in BP with no effect on HR or water intake. Results demonstrate the use of the RNA interference method in site-directed silencing of gene expression and provide a method for the in vivo study of Ang AT1 receptor function.
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PMID:Adenovirus-mediated small-interference RNA for in vivo silencing of angiotensin AT1a receptors in mouse brain. 1638 May 14