Tail-labelling of DNA probes using modified deoxynucleotide triphosphates and terminal deoxynucleotidyl tranferase. Application in electrochemical DNA hybridization and protein-DNA binding assays

2011 ◽  
Vol 9 (5) ◽  
pp. 1366 ◽  
Author(s):  
Petra Horáková ◽  
Hana Macíčková-Cahová ◽  
Hana Pivoňková ◽  
Jan Špaček ◽  
Luděk Havran ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Hybridization ◽  
Dna Probes ◽  
Binding Assays

scholarly journals Novel Transcriptional Regulons for Autotrophic Cycle Genes in Crenarchaeota

2015 ◽  
Vol 197 (14) ◽  
pp. 2383-2391 ◽  
Author(s):  
Semen A. Leyn ◽  
Irina A. Rodionova ◽  
Xiaoqing Li ◽  
Dmitry A. Rodionov
Keyword(s):  
Carbon Dioxide ◽  
Transcriptional Regulation ◽  
Comparative Genomics ◽  
Dna Binding ◽  
Transcriptional Control ◽  
Binding Assays ◽  
Promoter Regions ◽  
Content Type ◽  
Genome Context

ABSTRACTAutotrophic microorganisms are able to utilize carbon dioxide as their only carbon source, or, alternatively, many of them can grow heterotrophically on organics. Different variants of autotrophic pathways have been identified in various lineages of the phylumCrenarchaeota. Aerobic members of the orderSulfolobalesutilize the hydroxypropionate-hydroxybutyrate cycle (HHC) to fix inorganic carbon, whereas anaerobicThermoprotealesuse the dicarboxylate-hydroxybutyrate cycle (DHC). Knowledge of transcriptional regulation of autotrophic pathways inArchaeais limited. We applied a comparative genomics approach to predict novel autotrophic regulons in theCrenarchaeota. We report identification of two novel DNA motifs associated with the autotrophic pathway genes in theSulfolobales(HHC box) andThermoproteales(DHC box). Based on genome context evidence, the HHC box regulon was attributed to a novel transcription factor from the TrmB family named HhcR. Orthologs of HhcR are present in allSulfolobalesgenomes but were not found in other lineages. A predicted HHC box regulatory motif was confirmed byin vitrobinding assays with the recombinant HhcR protein fromMetallosphaera yellowstonensis. For the DHC box regulon, we assigned a different potential regulator, named DhcR, which is restricted to the orderThermoproteales. DhcR inThermoproteus neutrophilus(Tneu_0751) was previously identified as a DNA-binding protein with high affinity for the promoter regions of two autotrophic operons. The global HhcR and DhcR regulons reconstructed by comparative genomics were reconciled with available omics data inMetallosphaeraandThermoproteusspp. The identified regulons constitute two novel mechanisms for transcriptional control of autotrophic pathways in theCrenarchaeota.IMPORTANCELittle is known about transcriptional regulation of carbon dioxide fixation pathways inArchaea. We previously applied the comparative genomics approach for reconstruction of DtxR family regulons in diverse lineages ofArchaea. Here, we utilize similar computational approaches to identify novel regulatory motifs for genes that are autotrophically induced in microorganisms from two lineages ofCrenarchaeotaand to reconstruct the respective regulons. The predicted novel regulons in archaeal genomes control the majority of autotrophic pathway genes and also other carbon and energy metabolism genes. The HhcR regulon was experimentally validated by DNA-binding assays inMetallosphaeraspp. Novel regulons described for the first time in this work provide a basis for understanding the mechanisms of transcriptional regulation of autotrophic pathways inArchaea.

scholarly journals Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression

2021 ◽  
Vol 9 (1) ◽  
pp. 6
Author(s):  
Narendra Pratap Singh ◽  
Bony De Kumar ◽  
Ariel Paulson ◽  
Mark E. Parrish ◽  
Carrie Scott ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Embryonic Stem ◽  
Binding Motif ◽  
Binding Assays ◽  
Histone Marks ◽  
Genome Wide ◽  
Hox Cofactors

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

Genomic variability within laboratory and wild subspecies of Heligmosomoides polygyrus

1994 ◽  
Vol 68 (2) ◽  
pp. 93-96 ◽  
Author(s):  
M.A. Abu-Madi ◽  
A.P. Reid ◽  
J.W. Lewis ◽  
W.M. Hominick
Keyword(s):  
Restriction Endonuclease ◽  
Ribosomal Dna ◽  
Dna Hybridization ◽  
Genomic Dna ◽  
Dna Probes ◽  
Heligmosomoides Polygyrus ◽  
Banding Patterns ◽  
Genomic Variability

AbstractGenomic DNA extracted from laboratory and wild subspecies of the trichostrongyle nematode Heligmosomoides polygyrus were compared using RFLP and DNA/DNA hybridization techniques. Eight restriction endonuclease digests of the genomic DNA of the two subspecies were hybridized with heterologous ribosomal DNA probes and the total radio-isotope labelled DNA of the laboratory subspecies. DNA hybridization of the two subspecies of H. polygyrus yielded different banding patterns when probed with the rDNA clones in Pvu II digests and when total genomic DNA was used as the probe in Hind III and Pvu II digests. The remaining hybridization profiles of both subspecies were identical.

The snail repressor establishes a muscle/notochord boundary in the Ciona embryo

Development ◽  
1998 ◽  
Vol 125 (13) ◽  
pp. 2511-2520 ◽  
Author(s):  
S. Fujiwara ◽  
J.C. Corbo ◽  
M. Levine
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Sites ◽  
Promoter Region ◽  
Muscle Cells ◽  
Specific Pattern ◽  
Binding Assays ◽  
Heterologous Promoter ◽  
Present Evidence ◽  
Tail Muscle

Previous studies have identified a minimal 434 bp enhancer from the promoter region of the Ciona Brachyury gene (Ci-Bra), which is sufficient to direct a notochord-specific pattern of gene expression. Here we present evidence that a Ciona homolog of snail (Ci-sna) encodes a repressor of the Ci-Bra enhancer in the tail muscles. DNA-binding assays identified four Ci-Sna-binding sites in the Ci-Bra enhancer, and mutations in these sites cause otherwise normal Ci-Bra/lacZ transgenes to be misexpressed in ectopic tissues, particularly the tail muscles. Selective misexpression of Ci-sna using a heterologous promoter results in the repression of Ci-Bra/lacZ transgenes in the notochord. Moreover, the conversion of the Ci-Sna repressor into an activator results in the ectopic induction of Ci-Bra/lacZ transgenes in the muscles, and also causes an intermixing of notochord and muscle cells during tail morphogenesis. These results suggest that Ci-Sna functions as a boundary repressor, which subdivides the mesoderm into separate notochord and tail muscle lineages.

Nucleotides bearing aminophenyl- or aminonaphthyl-3-methoxychromone solvatochromic fluorophores for enzymatic construction of DNA probes for detection of protein-DNA binding

2021 ◽  
Author(s):  
Jan Matyasovsky ◽  
Laure Tack ◽  
Attila Palagyi ◽  
Miroslav Kuba ◽  
Radek Pohl ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Probes ◽  
Nucleoside Triphosphates

We designed and synthesized nucleosides bearing aminophenyl- or aminonaphthyl-3-methoxychromone fluorophores attached at position 5 of cytosine or thymine and converted them to nucleoside triphosphates. The fluorophores exerted solvatochromic fluorescence with...

Examination of colonies and stool blots for detection of enteropathogens by DNA hybridization with eight DNA probes.

1989 ◽  
Vol 27 (2) ◽  
pp. 331-334 ◽  
Author(s):  
P Echeverria ◽  
D N Taylor ◽  
J Seriwatana ◽  
J E Brown ◽  
U Lexomboon
Keyword(s):  
Dna Hybridization ◽  
Dna Probes

scholarly journals E4F and ATF, two transcription factors that recognize the same site, can be distinguished both physically and functionally: a role for E4F in E1A trans activation.

1990 ◽  
Vol 10 (10) ◽  
pp. 5138-5149 ◽  
Author(s):  
R J Rooney ◽  
P Raychaudhuri ◽  
J R Nevins
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Stable Complex ◽  
Small Subset ◽  
Adenovirus Infection ◽  
Binding Assays ◽  
Dna Binding Activity ◽  
Sequence Recognition ◽  
Nuclear Factors ◽  
Dna Binding Factors

Previous experiments have identified an element in the adenovirus E4 promoter that is critical for E1A-dependent trans activation and that can confer inducibility to a heterologous promoter. This DNA element is a recognition site for multiple nuclear factors, including ATF, which is likely a family of DNA-binding factors with similar DNA recognition properties. However, ATF activity was found not to be altered in any demonstrable way as a result of adenovirus infection. In contrast, another factor that recognizes this element, termed E4F, was found at only very low levels in uninfected cells but was increased markedly upon adenovirus infection, as measured in DNA-binding assays. Although both the ATF activity and the E4F activity recognized and bound to the same two sites in the E4 promoter, they differed in their sequence recognition of these sites. Furthermore, E4F bound only to a small subset of the ATF recognition sites; for instance, E4F did not recognize the ATF sites in the E2 or E3 promoters. Various E4F and ATF binding sites were inserted into an expression vector and tested by cotransfection assays for responsiveness to E1A. We found that a sequence capable of binding E4F could confer E1A inducibility. In contrast, a sequence that could bind ATF but not E4F did not confer E1A inducibility. We also found that E4F formed a stable complex with the E4 promoter, whereas the ATF DNA complex was unstable and rapidly dissociated. We conclude that the DNA-binding specificity of E4F as well as the alterations in DNA-binding activity of E4F closely correlates with E1A stimulation of the E4 promoter.

scholarly journals Dibenzopyridoimidazocinnolinium cations: a new family of light-up fluorescent DNA probes

2018 ◽  
Vol 5 (12) ◽  
pp. 1916-1927 ◽  
Author(s):  
Pedro Bosch ◽  
David Sucunza ◽  
Francisco Mendicuti ◽  
Alberto Domingo ◽  
Juan J. Vaquero
Keyword(s):  
Dna Binding ◽  
Fluorescence Intensity ◽  
Living Cells ◽  
Dna Probes ◽  
Live Cell ◽  
Binding Ability ◽  
Active Uptake ◽  
New Family ◽  
Cell Staining ◽  
New Compounds

A new family of weakly fluorescent azonia cations with DNA-binding ability by intercalation whose fluorescence intensity increases significantly upon DNA addition is reported. A live-cell staining cells analysis showed the capacity of these new compounds for active uptake and accumulation by living cells.

scholarly journals Conservation of noIR in the Sinorhizobium and Rhizobium Genera of the Rhizobiaceae Family

1998 ◽  
Vol 11 (12) ◽  
pp. 1186-1195 ◽  
Author(s):  
Ernö Kiss ◽  
Peter Mergaert ◽  
Boglàrka Olàh ◽  
Attila Kereszt ◽  
Christian Staehelin ◽  
...  
Keyword(s):  
Dna Binding ◽  
Primary Structure ◽  
Dna Hybridization ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Leguminosarum ◽  
Negative Regulation ◽  
Regulatory Proteins ◽  
Dna Binding Domain ◽  
Nod Factor ◽  
Homologous Sequences

In Sinorhizobium meliloti the NolR repressor displays differential negative regulation of nodulation genes and is required for optimal nodulation. Here, we demonstrate that the NolR function is not unique to S. meliloti but is also present in other species of the Rhizobiaceae family. DNA hybridization indicates the presence of nolR homologous sequences in species belonging to the Rhizobium and Sinorhizobium genera while no hybridization signal was detected in species from the Mesorhizobium, Bradyrhizo-bium, Azorhizobium, and Agrobacterium genera. We isolated the nolR gene from the Rhizobium leguminosarum bv. viciae strain TOM and showed that the TOM nolR gene acts similarly to S. meliloti nolR by repressing the expression of both the nodABCIJ and the nodD genes, resulting in decreased Nod factor production. The presence of a functional nolR gene in R. leguminosarum is correlated with an increased rate and extent of nodulation of pea. The conserved primary structure, the location of the DNA-binding domain, and the similar size of NolR proteins, compared with a family of small bacterial regulatory proteins including HlyU, SmtB, and the ArsR-type regulators, revealed that NolR belongs to this family.

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