Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Retrotransposons have clearly molded the structure of the human genome. The reverse transcriptase coded for by long interspersed nuclear elements (LINEs) accounts for 35% of the human genome, with 8-9 x 10(5) copies of the most common human LINE element, L1Hs. Retrotransposons cycle through an RNA intermediate with transcription as the rate limiting step. Because various retrotransposons have been demonstrated to be induced by environmental stimuli, we investigated the response of the L1Hs promoter to various agents. L1Hs promoter activity was analyzed by transfecting an L1Hs-expressing cell line with plasmids containing one of two L1Hs promoters fused to the LacZ reporter gene. L1Hs promoter activity was then monitored with a beta-galactosidase assay. Treatment with UV light and heat shock resulted in a small increase in beta-galactosidase activity from one promoter, while treatment with tetradecanoylphorbol 13-acetate resulted in small increases in beta-galactosidase activity from both promoters. No increase in beta-galactosidase activity was observed after exposure to X-rays or hydrogen peroxide.
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PMID:Environmental factors affecting transcription of the human L1 retrotransposon. II. Stressors. 1262 Oct 71

The effect of reverse transcriptase (RT) catalyzed mutations on continued extension of the nascent DNA chain was investigated. A system using the alpha-lac gene of beta-galactosidase as template and two sets of conditions was used. In one, RT was allowed to reassociate with the primer-template after falling off, while in a second RT was sequestered after dissociating. In the first condition, subsequent extension of errors that may have initially caused enzyme dissociation can occur. In the second, such errors would not be extended. Fully extended products were assayed by alpha-complementation to assess mutation frequency. A lower frequency in the latter scenario implies that some errors caused the polymerase to dissociate. Allowing only a single binding event lowered the mutation frequency of the products by about 1/2 suggesting that approximately 1 in 2 errors terminated synthesis. In other experiments, when added to a primer-template with a terminal mismatch at the 3' end, RT dissociated from the template about 50-90% of the time (depending on mismatch type) rather than extending. Running start reactions indicated that extension was more likely if an actively synthesizing RT made the mutation. RT RNase H cleavage analysis showed that 3' mismatches weakened the association of RT with the primer-terminus. Taken together, these results suggest that an actively synthesizing RT enzyme that has just made a mistake is likely bound in a configuration that generally enhances extension of the mistake. This is in contrast to RTs that must bind to then extend mismatches. The importance of these findings with respect to the mechanism of mutagenesis is discussed.
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PMID:Analysis of mutations made during active synthesis or extension of mismatched substrates further define the mechanism of HIV-RT mutagenesis. 1274 51

The authors have derived a new beta-cell line (betaIns2(-/-lacZ)) from Ins2-/- mice that carry the lacZ reporter gene under control of the Ins2 promoter. betaIns2(-/-lacZ) cells stained positively using anti-insulin antibody, expressed beta-cell-specific genes encoding the transcription factor PDX-1, glucokinase, and Glut-2, retained glucose-responsiveness for insulin secretion, and expressed the lacZ gene. Analysis of Ins1 expression by reverse transcriptase-polymerase chain reaction (RT-PCR) showed that Ins1 transcripts were significantly raised to compensate for the lack of Ins2 transcripts in betaIns2(-/-lacZ) cells, as compared to those found in betaTC1 cells expressing both Ins1/Ins2. Thus, transcriptional up-regulation of the remaining functional insulin gene in Ins2-/- mice could potentially contribute to the beta-cell adaptation exhibited by these mutants, in addition to the increase in beta-cell mass that we previously reported. We have also shown that lacZ expression, as analyzed by determining beta-galactosidase activity, was up-regulated by incubating betaIns2(-/-lacZ) cells with GLP-1 and/or IBMX, 2 known stimulators of insulin gene expression. These cells thus represent a new tool for testing of molecules capable of stimulating Ins2 promoter activity.
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PMID:Ins1 gene up-regulated in a beta-cell line derived from Ins2 knockout mice. 1274 65

Here, we show that inhibition of c-Myc causes a proliferative arrest of M14 melanoma cells through cellular crisis, evident by the increase in size, multiple nuclei, vacuolated cytoplasm, induction of senescence-associated beta-galactosidase activity and massive apoptosis. The c-Myc-induced crisis is associated with decreased human telomerase reverse transcriptase expression, telomerase activity, progressive telomere shortening, glutathione (GSH), depletion and, increased production of reactive oxygen species. Treatment of control cells with L-buthionine sulfoximine decreases GSH to levels of c-Myc low expressing cells, but it does not modify the growth kinetic of the cells. Surprisingly, when GSH is increased in the c-Myc low expressing cells by treatment with N-acetyl-L-cysteine, cells escape crisis. To test the hypothesis that both oxidative stress and telomerase dysfunction are involved in the c-Myc-dependent crisis, we directly inhibited telomerase function and glutathione levels. Inactivation of telomerase, by expression of a catalytically inactive, dominant negative form of reverse transcriptase, reduces cellular lifespan by inducing telomere shortening. Treatment of cells with L-buthionine sulfoximine decreases GSH content and accelerates cell crisis. Analysis of telomere status demonstrated that oxidative stress affects c-Myc-induced crisis by increasing telomere dysfunction. Our results demonstrate that inhibition of c-Myc oncoprotein induces cellular crisis through cooperation between telomerase dysfunction and oxidative stress.
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PMID:Inhibition of c-Myc oncoprotein limits the growth of human melanoma cells by inducing cellular crisis. 1282 59

Integration of IS1301 into an AT-rich inverted repeat located upstream of the ltx operon was previously shown to confer a hyperleukotoxic phenotype in Actinobacillus actinomycetemcomitans IS1 (T. He, T. Nishihara, D. R. Demuth, and I. Ishikawa, J. Periodontol. 70:1261-1268, 1999), but the mechanism leading to increased leukotoxin production was not determined. We show that an IS1 ltx promoter::lacZ reporter construct expresses 12-fold higher levels of beta-galactosidase activity than a reporter containing the ltx promoter from A. actinomycetemcomitans 652, suggesting that IS1301 increases transcription of the ltx operon. Examination of the IS1301 sequence identified a potential outwardly directed promoter. However, site-specific mutagenesis of the -35 element of the putative promoter had no effect on the transcriptional activity of the IS1 reporter construct. Furthermore, reverse transcriptase PCR and real-time PCR experiments did not detect a transcript that was initiated within IS1301. These results suggest that increased expression of leukotoxin in strain IS1 does not arise from an outwardly directed IS1301 promoter. To determine how IS1301 alters transcriptional regulation of the ltx operon, cis-acting sequences that regulate leukotoxin expression were identified. The AT-rich sequence that resides downstream from the site of IS1301 insertion was shown to function as a positive cis-acting regulator of leukotoxin expression. This sequence resembles an UP element in its location, AT-rich content, and activity and is homologous to the consensus UP element sequence. In addition, a negative cis-acting sequence was identified upstream from the site of IS1301 insertion, and deletion of this region increased promoter activity by fourfold. Mobility shift experiments showed that this region bound to a protein(s) in extracts from A. actinomycetemcomitans 652. The specific sequences required for this interaction were localized to a 26-nucleotide segment of the ltx promoter that resides 17 bp upstream from the site of IS1301 insertion. Together, these results suggest that positive and negative cis-acting sequences regulate leukotoxin expression and that IS1301 may increase transcription of the ltx operon in A. actinomycetemcomitans IS1 by displacing a negative cis-acting regulator approximately 900 bp upstream from the basal elements of the ltx promoter.
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PMID:Positive and negative cis-acting regulatory sequences control expression of leukotoxin in Actinobacillus actinomycetemcomitans 652. 1450 Apr 84

Mature spermatozoa spontaneously take up foreign DNA molecules which can be delivered to embryos at fertilization. Recently we discovered an endogenous reverse transcriptase (RT) activity in mouse spermatozoa which can reverse-transcribe exogenous RNA molecules into cDNA copies. Here we have sought to establish whether foreign RNA is a suitable substrate for the sperm RT to generate new functional genes. In vitro fertilization (IVF) experiments were carried out with spermatozoa that were preincubated with RNA from hybrid murine leukemia virus/virus-like 30S (MLV/VL30) beta-galactosidase (beta-gal) gene-containing vector. The RNA was taken up by sperm cells, reverse-transcribed, delivered to embryos upon IVF, and propagated in a mosaic pattern in founders and further in the F1 progeny. beta-gal protein expression was detected in several tissues from both F0 and F1 animals. These results indicate that spermatozoa can reverse-transcribe exogenous RNA so as to generate transcriptionally competent sequences that are transmitted to offspring upon fertilization.
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PMID:Sperm endogenous reverse transcriptase as mediator of new genetic information. 1465 76

Gene therapy is emerging as a realistic addition to the therapeutic arsenal in heart failure, but the search for suitable vectors for cardiac transfection is still ongoing. In this study, we explore the applicability of recombinant Semliki Forest virus (SFV) in heart failure. SFV was intracoronarily delivered 2 weeks after induction of myocardial infarction in the rat model for heart failure. Duration of SFV expression was determined, and tissue distribution was studied by histochemical, biochemical, and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. Expression of SFV-mediated transfection in the heart reached its maximum after 48-72 h and subsided within a week. Intracoronary administration of SFV efficiently transfected the non-infarcted cardiac wall, resulting in high levels of beta-galactosidase (beta-gal) activity (1337 +/- 537 IU/mg) and lacZ RNA in the hearts of all rats, whereas brain, kidney, liver, lung, spleen, and testis were lacZ negative. In conclusion, intracoronarily delivered SFV has a favourable distribution pattern, showing expression of the transgene restricted to the heart.
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PMID:Semliki Forest virus is an efficient and selective vector for gene delivery in infarcted rat heart. 1524 44

The RNase H primer grip of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) contacts the DNA primer strand and positions the template strand near the RNase H active site, influencing RNase H cleavage efficiency and specificity. Sequence alignments show that 6 of the 11 residues that constitute the RNase H primer grip have functional equivalents in murine leukemia virus (MLV) RT. We previously showed that a Y586F substitution in the MLV RNase H primer grip resulted in a 17-fold increase in substitutions within 18 nucleotides of adenine-thymine tracts, which are associated with a bent DNA conformation. To further determine the effects of the MLV RNase H primer grip on replication fidelity and viral replication, we performed additional mutational analysis. Using either beta-galactosidase (lacZ) or green fluorescent protein (GFP) reporter genes, we found that S557A, A558V, and Q559L substitutions resulted in statistically significant increases in viral mutation rates, ranging from 2.1- to 3.8-fold. DNA sequencing analysis of nonfluorescent GFP clones indicated that the mutations in RNase H primer grip significantly increased the frequency of deletions between the primer-binding site (PBS) and sequences downstream of the PBS. In addition, quantitative real-time PCR analysis of reverse transcription products revealed that the mutant RTs were substantially inefficient in plus-strand DNA transfer relative to the wild-type control. These results indicate that the MLV RNase H primer grip is an important determinant of in vivo fidelity of DNA synthesis and suggest that the mutant RT was unable to copy through the DNA-RNA junction of the minus-strand DNA and the tRNA because of its bent conformation resulting in error-prone plus-strand DNA transfer.
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PMID:Mutations in the RNase H primer grip domain of murine leukemia virus reverse transcriptase decrease efficiency and accuracy of plus-strand DNA transfer. 1559 35

Fetal cardiomyocytes have been proposed as a potential source of cell-based therapy for heart failure. This study examined cellular senescence in cultured human fetal ventricular cardiomyocytes (HFCs). HFCs were isolated and identified by immunocytochemistry and RT-PCR. Cells were found to senesce after 20-25 population doublings, as determined by growth arrest, morphological changes and senescence-associated beta-galactosidase activity. Using the telomeric repeat amplification protocol assay, telomerase activity was undetectable in primary HFCs. Cells were transduced to express the human reverse transcriptase subunit (hTERT) of telomerase. This resulted in greatly increased telomerase activity, but no significant lifespan extension. Analysis of telomere length in primary HFCs revealed that the senescent phenotype was not accompanied by telomere shortening. Telomeres in hTERT-positive cells were elongated in comparison with primary cells, and elongation was retained in senescent cells. Levels of the tumor suppressor protein p16INK4A increased in all senescent cells whether telomerase-positive or -negative. Senescence was accompanied by a decline in transcript levels of the polycomb gene Bmi-1, Ets1 and Ets2 transcription factors, and Id1, Id2 and Id3 helix-loop-helix proteins, suggesting roles for these genes in maintenance of cardiomyocyte proliferative capacity. In addition to offering novel insights into the behavior of human fetal cardiomyocytes in culture, these findings have implications for the development of a cell-based therapy for cardiac injury using primary fetal heart tissue.
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PMID:Telomere-independent cellular senescence in human fetal cardiomyocytes. 1565 10

Citrus unshiu is freeze tolerant to -10 degrees C when fully acclimated after exposure to cold, nonfreezing temperatures. To gain an understanding of its cold tolerance mechanism, mRNA differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) and quantitative relative RT-PCR were used to study gene expression under a gradual cold-acclimation temperature regime. Six up-regulated and two down regulated genes were identified based on their amino acid sequences. The identified proteins encoded by the up-regulated genes were: 14-3-3 protein, 40S ribosomal protein S23, putative 60S ribosomal protein L15, nucleoside diphosphate kinase III protein, regulator of chromosome condensation-like protein, and amino acid permease 6. The proteins encoded by the two down-regulated genes were: miraculin-like protein and beta-galactosidase. Their individual function has been briefly reviewed based on published information. In addition to the findings in this study, we compared the function of cold responsive genes of Poncirus trifoliata, a very cold hardy relative of Citrus species that is freeze tolerant to -30 degrees C when fully acclimated, to the function of genes in the current study.
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PMID:Identification of cold acclimated genes in leaves of Citrus unshiu by mRNA differential display. 1612 77


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