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

A 100-kDa DNA binding protein was found to be dramatically up-regulated upon the mitogenic stimulation of murine splenocytes with bacterial lipopolysaccharide (LPS). The induced DNA binding protein was also found to exhibit moderate binding specificity for the immunoglobulin isotype switch DNA repeats. Furthermore, the induction of the 100-kDa protein by LPS was found to be mediated by both an increase in the protein's stability and an increase in the synthesis of the protein. In vitro phosphorylation experiments revealed that the 100-kDa DNA binding protein was one of the most heavily phosphorylated proteins in both lymphoid and nonlymphoid nuclear extracts. Although this in vitro phosphorylation initially appeared to be mediated by a potent nuclear kinase activity, it was later determined that a significant part of the detected labeling was due to the direct binding of ATP by the 100-kDa protein. Antibodies raised to the 100-kDa DNA binding protein were used to isolate cDNA clones from a lymphocyte cDNA lambda gt11 expression library. Nucleotide sequence analysis revealed that the cloned cDNAs were identical to the mouse nucleolin gene. The beta-galactosidase fusion proteins (encoded by exons 3-14 of nucleolin) and a more severely truncated 45-kDa protein (encoded by exons 5-14 of nucleolin) were both found to bind strongly to DNA and ATP. Furthermore, the strength of DNA binding was found to be highly dependent on the overall dG content of the DNA probes. Our experiments also revealed that apart from binding ATP and G-rich DNA, nucleolin directly bound GTP, dATP, and dGTP, but not dCTP, dTTP, or dUTP. Computer analysis revealed that the putative ATP binding domains appear to fall within two of the phylogenetically conserved RNA binding domains of nucleolin.
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PMID:The murine nucleolin protein is an inducible DNA and ATP binding protein which is readily detected in nuclear extracts of lipopolysaccharide-treated splenocytes. 769 29

The lacI gene has been used as a target gene in various mutation assays. We modified single strand conformation polymorphism (SSCP) analysis by introducing restriction digestion to detect mutations in the gene rapidly, and determined the sensitivity of the method. The entire coding sequence and partial promoter region of the lacI gene were amplified by the polymerase chain reaction with [alpha-32P]dCTP in a 1247 base pair fragment, digested into eight restriction fragments, and analyzed by SSCP. The sensitivity of the method was assessed using 160 phages with lacI mutations, which were selected by assay of expression of beta-galactosidase after their infection into E. coli. Of the 160 mutants, 146 (91.3%) showed shifted bands in the first condition of SSCP analysis (without glycerol, 20 degrees C). The remaining 14 mutants were analyzed in a second condition (with 5% glycerol, 20 degrees C), and eight of them showed shifted bands (cumulatively 96.3% of the 160 mutants). The remaining six mutants were analyzed in a third condition (with 5% glycerol, 10 degrees C), and all of them showed shifted bands (cumulatively 100%). Sequencing of the restriction fragments with mobility shifts in the 160 mutants revealed 108 kinds of mutations, 100 (92.6%) being detected in the first condition, seven (cumulatively 99.1%) in the second condition, and one (cumulatively 100%) in the third condition. This method greatly reduced the time to identify lacI mutations, and allowed the detection of multiple mutations in one lacI mutant. The results also show that in general PCR-SSCP analysis is very sensitive when test fragments are shorter than about 250 base pairs and electrophoresis is performed under at least two conditions.
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PMID:A rapid method for detection of mutations in the lacI gene using PCR-single strand conformation polymorphism analysis: demonstration of its high sensitivity. 775 92

For the purpose of studying the factors that cause wide variation in transient transgene expression in individual fish, a lacZ reporter gene linked to a carp beta-actin regulatory sequence was introduced into zebrafish embryos. As a general trend, a correlation between the number of transgene copies injected and the level of transgene expression was found. However, a substantial variation in the level of expression still occurred that could not be attributed to technical factors such as the difference in injected volume of the transgene. Co-injection of 32P-dCTP and transgene into the same embryo followed by detection of beta-galactosidase activity, has shown that the volume used for transgene injection, which was determined in terms of radioactivity, is not closely related to the level and location of transgene expression. Injection into the animal pole at zygote stage and the yolk cytoplasmic layer (YCL) at the 64-cell stage followed by determination of transgene expression in terms of unit injection volume, revealed that there are marked differences among tissues with regard to their capacity for transgene expression, and that the yolk syncytial layer is higher in this capacity. This high activity is assumed to be due to the high transcriptional activity or enhanced transgene replication in the syncytial layer, which is known to contain giant polyploid nuclei. The high levels of expression in the YSL may influence transient expression studies using quantitative comparative analyses and should be taken into consideration when expression data are derived from homogenates of yolk sac embryos.
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PMID:High transgene activity in the yolk syncytial layer affects quantitative transient expression assays in zebrafish Danio rerio) embryos. 884 May 26

The effects of deoxynucleoside triphosphate (dNTP) imbalances on the fidelity of human immunodeficiency virus type 1 (HIV-1) replication were investigated. Using detergent permeabilized virions and biased dNTP concentrations different types of hypermutants were readily produced. However, the mutant spectrum was different from naturally occurring hypermutants demonstrating that the host cell may restrict variation. Using a genetic screen based on the blue/white beta-galactosidase complementation assay, G --> A hypermutants were recovered from HIV-infected thymidine treated U937 cells. Furthermore, hypermutants were recovered from 1 to 2% of resting or activated peripheral blood mononuclear cells indicating that small proportions of primary cells had distorted intracellular [dTTP] and [dCTP]. Such imbalances may underlie a proportion of somatic and germline point mutations and shape to some extent the evolution of mammalian and viral genomes.
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PMID:HIV genetic variation is directed and restricted by DNA precursor availability. 923 17

A genetic screen based on the blue-white beta-galactosidase complementation assay designed to detect G-->A mutations arising during RNA-dependent DNA synthesis was used to compare the fidelity of mutant human immunodeficiency virus type 1 reverse transcriptases (RTs) with the mutations M230L and M230I with the wild-type enzyme, in the presence of biased deoxynucleoside triphosphate (dNTP) pools. The mutant RTs with the M230L and M230I changes were found to be 20 to 70 times less faithful than the wild-type RT in the presence of low [dCTP]/[dTTP] ratios but showed similar fidelity in assays carried out with equimolar concentrations of each nucleotide. Biased dNTP pools led to short tandem repeat deletions in the target sequence, which were also detectable with the assay. However, deletion frequencies were similar for all of the RTs tested. The reported data suggest that RT pausing due to the low dNTP levels available in the RT reaction mixture facilitates strand transfer, in a process that is not necessarily mediated by nucleotide misinsertion.
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PMID:Increased G-->A transition frequencies displayed by primer grip mutants of human immunodeficiency virus type 1 reverse transcriptase. 1469 33