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)

The MutS, MutL, and MutH proteins play major roles in several DNA repair pathways. We previously reported that the cellular amounts of MutS and MutH decreased by as much as 10-fold in stationary-phase cultures. Consequently, we tested whether the amounts of MutS, MutL, and MutH were regulated by two global regulators, RpoS (sigma38) and Hfq (HF-I [putative RNA chaperone]), which are involved in stationary-phase transition. We report here that mutations in hfq and rpoS reversed the stationary-phase down-regulation of the amounts of MutS and MutH. hfq regulation of the amount of MutS in stationary-phase cultures was mediated by RpoS-dependent and -independent mechanisms, whereas hfq regulation of the amount of MutH was mediated only through RpoS. Consistent with this interpretation, the amount of MutS but not MutH was regulated by Hfq, but not RpoS, in exponentially growing cells. The amount of MutL remained unchanged in rpoS, hfq-1, and rpoS+, hfq+ strains in exponentially growing and stationary-phase cultures and served as a control. The beta-galactosidase activities of single-copy mutS-lacZ operon and gene fusions suggested that hfq regulates mutS posttranscriptionally in exponentially growing cultures. RNase T2 protection assays revealed increased amounts of mutS transcript that are attributed to increased mutS transcript stability in hfq-1 mutants. Lack of Hfq also increased the amounts and stabilities of transcripts initiated from P(miaA) and P1hfqHS, two of the promoters for hfq, suggesting autoregulation, but did not change the half-life of bulk mRNA. These results suggest that the amounts of MutS and MutH may be adjusted in cells subjected to different stress conditions by an RpoS-dependent mechanism. In addition, Hfq directly or indirectly regulates several genes, including mutS, hfq, and miaA, by an RpoS-independent mechanism that destabilizes transcripts.
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PMID:Negative regulation of mutS and mutH repair gene expression by the Hfq and RpoS global regulators of Escherichia coli K-12. 939 14

The influence of actinomycin D on the synthesis of extracellular ribonucleases by "Bacillus intermedius" (binase), B.pumilus (RNAse Bp) and B.amyloliquefaciens (barnase) was studied comparatively. When added during the active synthesis of the enzymes actinomycin D stimulated the biosynthesis of binase and RNAse Bp and had no influence on the barnase biosynthesis. The response of the bacillary RNAse biosynthesis to the added actinomycin D correlated with the differences in the nucleotide sequences of the genes encoding the enzymes. The Escherichia coli SURE recombinant strains carrying the plasmids with the genes of binase, RNAse Bp and barnase under different regulatory sequences, as well as the E.coli MC4100 recombinant strains carrying the plasmids with the beta-galactosidase gene under the promoters of the bacillary RNAse were isolated. However, the expression of the bacillary ribonuclease genes in the E.coli recombinant strains carrying the plasmids with the genes of the enzymes, as well as the expression of the beta-galactosidase gene from the promotors of the bacillary RNAses was not stimulated by actinomycin D irrespective of the dose and addition time.
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PMID:[Actinomycin D influence on biosynthesis of extracellular ribonucleases by sporulating bacteria]. 946 96

Bacillus subtilis cytochrome c oxidase caa3 is encoded by the ctaCDEF genes at the ctaABCDEF locus, with the ctaBCDEF genes organized as an operon-like unit. A dyad symmetry sequence and a catabolite response element homolog can be recognized in the 240-bp intercistronic region between ctaB and ctaC. ctaB'-lacZ and ctaBCD'-lacZ transcriptional fusions integrated at the native locus were used to study catabolite effects on transcription of the ctaB and ctaCDEF genes. In Schaeffer's medium lacking glucose, ctaBCD'-lacZ was expressed at a very low level during the exponential phase, and expression increased about 30-fold 2 h after entry into the stationary phase. In the presence of 0.5% glucose, ctaBCD'-lacZ expression was totally repressed. In contrast to ctaBCD'-lacZ, ctaB'-lacZ was constitutively expressed regardless of carbon source. The ctaCDEF genes were separated from ctaB by insertion of plasmids carrying selectable markers in such a way that the ctaCDEF and ctaB transcription units remained intact. Enzymatic assays of caa3 with these constructs, showed that ctaCDEF was not expressed independently of ctaB. Also, when a 'ctaB-ctaC'-lacZ fusion (containing the ctaB-ctaC intercistronic region) was placed at a remote nonessential locus, beta-galactosidase activity could not be detected. The absence of a promoter in the ctaB-ctaC intercistronic space also was indicated by the inability to detect ctaC-specific transcripts with RNase protection assays, primer extension, and rapid amplification of 5' cDNA ends. Direct mRNA measurements showed that, in the presence of 0.5% glucose, ctaBCDEF transcripts terminated at the 3' end of the putative stem-loop structure and the distal portion was down-regulated. A possible mechanism for ctaCDEF gene regulation is suggested. Catabolite repression of ctaBCD'-lacZ was partly dependent on CcpA but was independent of HPr. The expression of ctaBCDEF also appears to require the strC, ctaA, and resD-resE gene products.
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PMID:Catabolite regulation of the Bacillus subtilis ctaBCDEF gene cluster. 982 23

Transgenic mice were generated expressing an alpha1 connexin/beta-galactosidase fusion protein previously shown to exert dominant negative effects on gap junctional communication. RNase protection analysis and assays for beta-galactosidase enzymatic activity showed that the transgene RNA and protein are expressed in the embryo and adult tissues. In situ hybridization analysis revealed that in the embryo, expression was predominantly restricted to neural crest cells and their progenitors in the dorsal neural tube, regions where the endogenous alpha1 connexin gene is also expressed. Dye-coupling analysis indicated that gap junctional communication was inhibited in the cardiac neural crest cells. All of the transgenic lines were homozygote inviable, dying neonatally and exhibiting heart malformations involving the right ventricular outflow tract-the same region affected in the alpha1 connexin knockout mice. As in the knockout mice, the conotruncal heart malformations were accompanied by outflow tract obstruction. Histological analysis showed that this was associated with abnormalities in the differentiation of the conotruncal myocardium. These results suggest that the precise level of gap junctional communication in cardiac neural crest cells is of critical importance in right ventricular outflow tract morphogenesis. Consistent with this possibility is the fact that cardiac crest cells from the alpha1 connexin knockout mice also exhibited a greatly reduced level of gap junctional communication. These studies show the efficacy of a dominant negative approach for manipulating gap junctional communication in the mouse embryo and demonstrate that targeted expression of this fusion protein can be a powerful tool for examining the role of gap junctions in mammalian development.
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PMID:Heart malformations in transgenic mice exhibiting dominant negative inhibition of gap junctional communication in neural crest cells. 985 55

Bmp2, a highly conserved member of the transforming growth factor-beta gene family, is crucial for normal development. Retinoic acid, combined with cAMP analogs, sharply induces the Bmp2 mRNA during the differentiation of F9 embryonal carcinoma cells into parietal endoderm. Retinoic acid (RA) also induces the Bmp2 gene in chick limb buds. Since normal Bmp2 expression may require an endogenous retinoid signal and aberrant Bmp2 expression may cause some aspects of RA-induced teratogenesis, we studied the mechanism underlying the induction of Bmp2. Measurements of the Bmp2 mRNA half-life and nuclear run-on assays indicated that RA stimulated the transcription rate of the Bmp2 gene. The results of ribonuclease protection and primer extension assays indicated that Bmp2 transcription started 2,127 nucleotides upstream of the translation start site in F9 cells. To identify genetic elements controlling this transcription rate increase, upstream and downstream genomic sequences flanking the Bmp2 gene were screened using chloramphenicol acetyltransferase reporter genes in F9 cells and beta-galactosidase reporter genes in Saccharomyces cerevisiae that were cotransformed with retinoic acid receptor and retinoid X receptor expression plasmids. RA-dependent transcriptional activation was detected between base pairs -2,373 and -2,316 relative to the translation start site. We also identified a required Sp1 binding site between -2,308 and -2,298. The data indicate that Bmp2 is directly regulated by retinoic acid-bound receptors and Sp1.
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PMID:Transcriptional regulation of the Bmp2 gene. Retinoic acid induction in F9 embryonal carcinoma cells and Saccharomyces cerevisiae. 988 May 12

High-performance liquid chromatography with on-line electrospray ionization mass spectrometry (ESI-LC/MS) was investigated for the analysis of carbohydrate heterogeneity using RNase B as a model glycoprotein. Oligosaccharides released from RNase B with endoglycosidase H were reduced and separated on a graphitized carbon column (GCC). GCC-HPLC/MS in the positive-ion mode was successful in the identification of one Man5GlcNAc, three Man6GlcNAc, three Man7GlcNAc, three Man8GlcNAc, one Man9GlcNAc, and an oligosaccharide having six hexose units (Hex) and two N-acetylhexosamine units (HexNAc). The branch structures of the three Man7GlcNAc isomers were determined by liquid chromatography with tandem mass spectrometry (LC/MS/MS). LC/MS/MS analysis was shown to be useful for the detection and identification of a trace amount of Hex6HexNAc2 alditol as a hybrid-type oligosaccharide. Its structure was confirmed by the combination of LC/MS with enzymatic digestion using beta-galactosidase and N-acetyl-beta-glucosaminidase. The relative quantities of high-mannose-type oligosaccharides in RNase B detected by ESI-LC/MS are in reasonable agreement with those by UV, high-pH anion-exchange chromatography with pulsed amperometric detection, fluorophore-assisted carbohydrate electrophoresis. Our results indicate that LC/MS and LC/MS/MS can be utilized to elucidate the distribution of oligosaccharides and their structures, which differ in molecular weight, sugar sequence, and branch structure.
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PMID:Analysis of carbohydrate heterogeneity in a glycoprotein using liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry. 1022 1

Three major inhibitors of the NF-kappaB/Rel family of transcription factors, IkappaBalpha, IkappaBbeta, and IkappaBepsilon, have been described. To examine the in vivo role of the most recently discovered member of the IkappaB family, IkappaBepsilon, we generated a null allele of the murine IkappaBepsilon gene by replacement of all coding sequences with nlslacZ. Unlike IkappaBalpha nullizygous mice, mice lacking IkappaBepsilon are viable, fertile, and indistinguishable from wild-type animals in appearance and histology. Analysis of beta-galactosidase expression pattern revealed that IkappaBepsilon is mainly expressed in T cells in the thymus, spleen, and lymph nodes. Flow cytometric analysis of immune cell populations from the bone marrow, thymus, spleen, and lymph nodes did not show any specific differences between the wild-type and the mutant mice, with the exception of a reproducible 50% reduction of the CD44-CD25+ T cell subspecies. The IkappaBepsilon-null mice present constitutive up-regulation of IgM and IgG1 Ig isotypes together with a further increased synthesis of these two isotypes after immunization against T cell-dependent or independent Ags. The failure of observable augmentation of constitutive nuclear NF-kappaB/Rel-binding activity is probably due to compensatory mechanisms involving IkappaBalpha and IkappaBbeta, which are up-regulated in several organs. RNase-mapping analysis indicated that IL-1alpha, IL-1beta, IL-1Ra, and IL-6 mRNA levels are constitutively elevated in thioglycolate-elicited IkappaBepsilon-null macrophages in contrast to GM-CSF, G-CSF, and IFN-gamma, which remain undetectable.
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PMID:IkappaBepsilon-deficient mice: reduction of one T cell precursor subspecies and enhanced Ig isotype switching and cytokine synthesis. 1057 Feb 87

To study the efficiency of RNA-based vaccines, RNA coding for the model antigen beta-galactosidase (beta-gal) was transcribed in vitro from a lacZ gene flanked by stabilizing Xenopus laevis beta-globin 5' and 3' sequences and was protected from RNase degradation by condensation with the polycationic peptide protamine. The liposome-encapsulated condensed RNA-peptide complex, the condensed RNA-peptide complex without liposome or naked, unprotected RNA, was injected into BALB/c (H-2(d)) mice. All preparations led to protein expression in the local tissue, activation of L(d)-restricted specific cytotoxic T lymphocytes (CTL) and production of IgG antibodies reactive against beta-gal. RNA-triggered CTL were as efficient in the lysis of lacZ-transfected target cells as CTL triggered by a lacZ-DNA eukaryotic expression vector. Immunization with RNA transcribed from a cDNA library from the beta-gal-expressing cell line P13.1 again led to beta-gal-specific CTL and IgG induction. Thus, both naked and protected RNA can be used to elicit a specific immune response in vivo, whereby the protected RNA is stable in vitro for a longer period of time. RNA vaccines can be produced in high amounts and have the same major advantages as DNA vaccines but lack the potentially harmful effect of DNA integration into the genome.
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PMID:In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies. 1060 21

Although the primary function of U1 snRNA is to define the 5' donor site of an intron, it can also block the accumulation of a specific RNA transcript when it binds to a donor sequence within its terminal exon. This work was initiated to investigate if this property of U1 snRNA could be exploited as an effective method for inactivating any target gene. The initial 10-bp segment of U1 snRNA, which is complementary to the 5' donor sequence, was modified to recognize various target mRNAs (chloramphenicol acetyltransferase [CAT], beta-galactosidase, or green fluorescent protein [GFP]). Transient cotransfection of reporter genes and appropriate U1 antitarget vectors resulted in >90% reduction of transgene expression. Numerous sites within the CAT transcript were suitable for targeting. The inhibitory effect of the U1 antitarget vector is directly related to the hybrid formed between the U1 vector and target transcripts and is dependent on an intact 70,000-molecular-weight binding domain within the U1 gene. The effect is long lasting when the target (CAT or GFP) and U1 antitarget construct are inserted into fibroblasts by stable transfection. Clonal cell lines derived from stable transfection with a pOB4GFP target construct and subsequently stably transfected with the U1 anti-GFP construct were selected. The degree to which GFP fluorescence was inhibited by U1 anti-GFP in the various clonal cell lines was assessed by fluorescence-activated cell sorter analysis. RNA analysis demonstrated reduction of the GFP mRNA in the nuclear and cytoplasmic compartment and proper 3' cleavage of the GFP residual transcript. An RNase protection strategy demonstrated that the transfected U1 antitarget RNA level varied between 1 to 8% of the endogenous U1 snRNA level. U1 antitarget vectors were demonstrated to have potential as effective inhibitors of gene expression in intact cells.
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PMID:Reduction of target gene expression by a modified U1 snRNA. 1128 60

We constructed two aptamers, each of which contains a 7-nt-long loop complementary to the anticodon loop of a suppressor tRNA. One of these aptamers can form a stable bimolecular complex with the suppressor tRNA in vitro and protects the 7 nt in the suppressor's anticodon loop from RNase S1. An Escherichia coli strain, carrying an amber mutation in the lac Z gene, produces beta-galactosidase only if the suppressor is present; the aptamer's coexpression in the cell inhibits the activity of the suppressor tRNA. Moreover, in E. coli extract, the aptamer partially inhibits the read-through of the stop codon on the part of the suppressor tRNA. These results point to a novel strategy that need not be limited to the suppressor tRNA. By constructing appropriate inducible aptamers, it may well be possible to effectively control translation in vivo.
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PMID:Trans-acting RNA inhibits tRNA suppressor activity in vivo. 1216 45


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