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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In vivo dimethyl sulfate footprinting of the Bacillus subtilis glnRA regulatory region under repressing and derepressing conditions demonstrated that the GlnR protein, encoded by glnR, interacts with two sites situated within and adjacent to the glnRA promoter. One site, glnRAo1, between positions -40 and -60 relative to the start point of transcription, is a 21-bp symmetrical element that has been identified as essential for glnRA regulation (H. J. Schreier, C. A. Rostkowski, J. F. Nomellini, and K. D. Hirschi, J. Mol. Biol. 220:241-253, 1991). The second site, glnRAo2, is a quasisymmetrical element having partial homology to glnRAo1 and is located within the promoter between positions -17 and -37. The symmetry and extent of modifications observed for each site during repression and derepression indicated that GlnR interacts with the glnRA regulatory region by binding to both sites in approximately the same manner. Experiments using potassium permanganate to probe open complex formation by RNA polymerase demonstrated that transcriptional initiation is inhibited by GlnR. Furthermore, distortion of the DNA helix within glnRAo2 occurred upon GlnR binding. While glutamine synthetase, encoded by glnA, has been implicated in controlling glnRA expression, analyses with dimethyl sulfate and potassium permanganate ruled out a role for glutamine synthetase in directly influencing transcription by binding to operator and promoter regions. Our results suggested that inhibition of transcription from the glnRA promoter involves GlnR occupancy at both glnRAo1 and glnRAo2. In addition, modification of bases within the glnRAo2 operator indicated that control of glnRA expression under nitrogen-limiting (derepressing) conditions included the involvement of a factor(s) other than GlnR.
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PMID:Interaction of the Bacillus subtilis glnRA repressor with operator and promoter sequences in vivo. 134 63

The Ig heavy chain (IgH) constant region (CH) class switch is manifested by DNA deletions which exchange the C mu gene of a functional VDJ-CH rearrangement for a C gamma, C epsilon or C alpha gene. Repetitive sequences (S regions) 5' of each CH gene mediate CH gene switch recombination by an illegitimate mechanism. S mu can be subdivided into S mu 5' (non-repetitive) and S mu 3' (repetitive) components with recombination occurring in either part. Here, we describe the properties of ubiquitous and B cell stage specific S mu binding factors NFS mu-U1 and NFS mu-B1 respectively. U1 only bound to S mu 5' sequences, and B1 to S mu 5', S mu 3' sequences and to other S regions with varying affinities. DMS and OP-Cu footprinting revealed the sequence AAAAAGCATGGCTGA in the U1 site while the B1 S mu 5' site overlapped the 3' end of the U1 binding site and also contained additional 3' flanking S mu repeat motifs (GAGCTGAGATGGGTGGGCT). Binding site competition assays reveal that NFS mu-B1 is either very related or identical to S alpha BP (described by Waters et al., Mol. Cell Biol. 9:5594, 1989) and BSAP (identified by Barberis et al., Genes Devl. 4:849, 1990) which were shown to bind to two sequences upstream of the S alpha repeats and within the promoters of sea urchin histone genes respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Properties of B cell stage specific and ubiquitous nuclear factors binding to immunoglobulin heavy chain gene switch regions. 141 57

The positions of interference points between the IclR repressor of the acetate operon of Escherichia coli and its specific operator were examined. The number and nature of nucleotides essential to repressor binding were determined by scanning populations of DNA previously methylated at guanine residues by dimethyl sulfate, or depurinated by treatment with formic acid, or depyrimidated by treatment with hydrazine. A total of 46 nucleotides, distributed almost equally between the two strands of the operator region, were found to be functionally important, although to a varying extent. These are clustered in two successive domains which expand from nucleotide -54 to nucleotide -27 and can organize in a palindrome-like structure containing a large proportion of A and T residues.
J Mol Biol 1992 Nov 05
PMID:Specific interactions between the IclR repressor of the acetate operon of Escherichia coli and its operator. 144 84

Dosage compensation of X-linked genes in male and female mammals is accomplished by random inactivation of one X chromosome in each female somatic cell. As a result, a transcriptionally active allele and a transcriptionally inactive allele of most X-linked genes reside within each female nucleus. To examine the mechanism responsible for maintaining this unique system of differential gene expression, we have analyzed the differential binding of regulatory proteins to the 5' region of the human hypoxanthine phosphoribosyltransferase (HPRT) gene on the active and inactive X chromosomes. Studies of DNA-protein interactions associated with the transcriptionally active and inactive HPRT alleles were carried out in intact cultured cells by in vivo footprinting by using ligation-mediated polymerase chain reaction and dimethyl sulfate. Analysis of the active allele demonstrates at least six footprinted regions, whereas no footprints were detected on the inactive allele. Of the footprints on the active allele, at least four occur over canonical GC boxes or Sp1 consensus binding sites, one is associated with a potential AP-2 binding site, and another is associated with a DNA sequence not previously reported to interact with a sequence-specific DNA-binding factor. While no footprints were observed for the HPRT gene on the inactive X chromosome, reactivation of the inactive allele with 5-azacytidine treatment restored the in vivo footprint pattern found on the active allele. Results of these experiments, in conjunction with recent studies on the X-linked human PGK-1 gene, bear implications for models of X chromosome inactivation.
Mol Cell Biol 1992 Dec
PMID:Multiple in vivo footprints are specific to the active allele of the X-linked human hypoxanthine phosphoribosyltransferase gene 5' region: implications for X chromosome inactivation. 144 69

We have previously shown that a trans-acting protein produced in some tissue culture cells positively control the transcriptional activity directed by the mouse p12 promoter. This nuclear protein exerts its positive activity by interacting with a regulatory sequence designated p12.A and located between the TATA and CCAAT box elements on the p12 gene promoter. Using DNase I and dimethyl sulfate methylation interference footprinting techniques coupled with gel retardation assays, we found evidence that the protein which binds to the p12.A element is the well-known transcription factor Sp1. Mutational analysis in transient transfection assays confirmed the positive activity exerted by this protein in every cell line tested. In agreement with this observation, we detected a p12.A-Sp1 binding activity in nuclear extracts prepared from all cell lines used. However, a similar binding activity could not be detected in a number of nuclear extracts prepared from normal mouse tissues. In this report, we provide the evidence that the lack of Sp1-binding activity results from the degradation of Sp1 in the kidney, liver, and pancreas of the mouse.
Mol Cell Biol 1992 Sep
PMID:Transcription of the mouse secretory protease inhibitor p12 gene is activated by the developmentally regulated positive transcription factor Sp1. 150 85

A major regulatory element required for expression of the human alpha-globin genes is located 40 kb upstream of the embryonic zeta-globin gene. To understand how this and other locus control region (LCR) elements contribute to high-level expression in erythroid cells, we have performed high-resolution, in vivo dimethyl sulfate footprinting. In addition, we have modified the dimethyl sulfate-based ligation-mediated polymerase chain reaction in vivo footprinting procedure to permit the assessment of interactions at guanine and adenine residues, rather than guanines alone. In vivo footprinting of the human alpha-LCR element carried on chromosome 16 in a mouse erythroleukemia cell environment revealed protein occupancy at GATA-1, AP-1/NF-E2, and CACC/GGTGG motifs, specific differences compared with in vitro protein binding, and distinct changes in one region upon dimethyl sulfoxide-induced cellular maturation. No protein contacts were detected in nonexpressing hepatoma cells. In addition, we have demonstrated that two AP-1 motifs in the alpha-LCR element which are occupied in vivo bind purified mouse NF-E2 protein in vitro. Our data suggest that three proteins, GATA-1, NF-E2, and unknown CACC/GGTGG factors, are minimally required as DNA-binding proteins for the function of LCR-like elements. The juxtaposition and interaction of these factors with each other, and with accessory proteins not directly in contact with DNA, are likely to account for the relative position independence of the upstream globin regulatory elements.
Mol Cell Biol 1992 May
PMID:In vivo footprinting of the human alpha-globin locus upstream regulatory element by guanine and adenine ligation-mediated polymerase chain reaction. 156 44

In this work we report on the isolation of an Escherichia coli K-12 mutation, which confers a high sensitivity to bacteria cells to mutagenesis by simple monofunctional alkylating agents. The mutation emerged spontaneously from a bacterial strain that already proved useful in various mutagenicity studies. By monitoring the influence of such a mutation on the frequency of induced mutation by ethylating (EMS, DES, ENU, ENNG) vs. methylating (MMS, DMS, MNU, MNNG) compounds, and on the in vivo repair capacity for different alkyl-DNA lesions (O6-alkG, N7-alkG, N3-meA), we conclude that the mutation should affect the gene (ogt) that encodes constitutive DNA repair alkyltransferase (ATase). Thus in the presence of ada, differences in mutagenicity were observed only with ethylating agents; the sensitization of cells to both the ethylating and methylating partners requiring, by contrast, the absence of the ada protein. These results support the reported in vitro substrate specificities for both ogt and ada ATases. The parental cells exhibited biphasic dose-response curves in accordance with the idea of low basal level saturation attributed to the uninducible ogt ATase. Deficient bacterial derivatives showed, by contrast, linear mutation induction responses. The in vivo removal of alkylated bases from DNA was measured in bacterial strains deficient in the excision repair pathway (delta uvrB) and unable to induce the adaptive response (ada::Tn10). The very low initial levels for O6-meG and O6-etG (1.1 and 0.2 molecules per cell, respectively) were readily repaired by the parental cells but remained unchanged in the hypermutable derivatives. This result suggests that in the absence of nucleotide excision repair and of the adaptive response, no alternative pathway, other than ogt, is available for the repair of the major mutagenic lesion, O6-alkG, at least during the first 4 hours after alkylation. Comparatively, no differences were found in the capacity to repair the major lethal adduct, N3-meA, in agreement with the fact that no effect on cell survival was detected. In conclusion, we propose that the biological significance of the ogt protein relies mainly on its ability to prevent mutagenesis by low levels of bulkier ethylation products (especially in the absence of uvr excision repair.(ABSTRACT TRUNCATED AT 400 WORDS)
Environ Mol Mutagen 1992
PMID:Mutagenesis and DNA repair for alkylation damages in Escherichia coli K-12. 160 Sep 55

The promoters of two interferon-induced genes (the ISG54 and guanylate-binding protein [GBP] genes) have been analyzed in whole cells and in isolated nuclei by using a new genomic sequencing technique. The ISG54 gene contains an interferon-simulating response element (ISRE), earlier shown to be necessary and sufficient for alpha interferon (IFN-alpha) induction, that appeared complexed with proteins in both transcribing and nontranscribing cells. However, the extent of protection and hypersensitivity to DNase I or dimethyl sulfate within the ISRE region was changed upon transcriptional induction, suggesting the binding of different factors in different transcriptional states. In addition to the ISRE, the GBP gene needs a newly recognized DNA element, called the GAS, that partly overlaps the ISRE for full induction by either IFN-alpha or IFN-gamma. This GAS element was transiently protected against DNase I in the nuclei of interferon-treated cells but was not protected at later times when transcription reached maximal levels. Thus, the GAS-binding activity may be necessary only transiently for the initial assembly of a transcription initiation complex on the GBP promoter. Dimethyl sulfate methylation of genomic DNA performed on intact cells showed a characteristic sensitivity over the GAS that correlated with transcription levels and that persisted longer than did DNase I protection over the GAS. These results demonstrate the involvement of the GAS in IFN-alpha and -gamma induction of GBP and suggest the presence of an altered DNA conformation or a small protein in the major groove of the GAS associated with ongoing GBP transcription.
Mol Cell Biol 1992 Jan
PMID:Interferon induction of gene transcription analyzed by in vivo footprinting. 172 91

PU.1 is a B-cell- and macrophage-specific transcription factor. By an electrophoretic mobility shift assay and dimethyl sulfate methylation interference assays, we show that PU.1 binds to DNA sequences within the immunoglobulin kappa 3' enhancer (kappa E3'). Binding of PU.1 to the kappa E3' enhancer assists the binding of a second tissue-restricted factor, NF-EM5, to an adjacent site. Binding of NF-EM5 to kappa E3' DNA sequences requires protein-protein interaction with PU.1 as well as specific protein-DNA interactions. This is the first known instance of PU.1 interacting with another cellular protein. NF-EM5 does not cofractionate with PU.1, suggesting that it is a distinct protein and is not a posttranslational modification of PU.1. UV-crosslinking studies and elution from sodium dodecyl sulfate-polyacrylamide gels indicate that NF-EM5 is a protein of approximately 46 kDa. Site-directed mutagenesis studies of the PU.1- and EM5-binding sites indicate that these sites play important roles in kappa E3' enhancer activity. By using a series of PU.1 deletion constructs, we have identified a region in PU.1 that is necessary for interaction with NF-EM5. This segment encompasses a 43-amino-acid region with PEST sequence homology, i.e., one that is rich in proline (P), glutamic acid (E), serine (S), and threonine (T).
Mol Cell Biol 1992 Jan
PMID:PU.1 recruits a second nuclear factor to a site important for immunoglobulin kappa 3' enhancer activity. 172 11

A method to investigate the structure of RNA molecules within intact plant tissues has been developed. The RNA structures are analyzed using dimethyl sulfate (DMS), which modifies substituents of adenine and cytosine residues within single-stranded regions of RNA molecules. Reactive sites are identified by primer extension analysis. Using this procedure, an analysis of the secondary structure of the cytoplasmic 18S ribosomal RNA in soybean seedling leaves has been completed. DMS modification data are in good agreement with the phylogenetic structure predicted for soybean 18S rRNA. However, there are a few notable exceptions where residues thought to be involved in double-stranded regions in all 18S rRNAs are strongly modified in soybean leaf samples. These data taken together with the phylogenetic structure suggest that alternate structures may exist in vivo. The further applicability of this technique is demonstrated by comparing the modification pattern obtained in vivo to that obtained in vitro for a particular mRNA molecule encoding the small subunit of ribulose-1,5-bisphosphate carboxylase. The results obtained are compared to a predicted minimum energy secondary structure. The data indicate that the conformation of RNA molecules within the cell may not be reflected in a structural analysis of purified mRNA molecules.
Plant Mol Biol 1992 Jan
PMID:In vivo analysis of plant RNA structure: soybean 18S ribosomal and ribulose-1,5-bisphosphate carboxylase small subunit RNAs. 173 85


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