Xrel3 is required for head development in Xenopus laevis

Development ◽  
2001 ◽  
Vol 128 (2) ◽  
pp. 263-273
Author(s):  
B.B. Lake ◽  
R. Ford ◽  
K.R. Kao
Keyword(s):  
Dna Sequences ◽  
Dorsal Side ◽  
Related Gene ◽  
Wild Type ◽  
Neural Patterning ◽  
Nf Kappa B ◽  
Head Development ◽  
Dorsal Aspect ◽  
Vitro Binding

The Rel/NF-kappa B gene family encodes a large group of transcriptional activators involved in myriad differentiation events, including embryonic development. We have shown previously that Xrel3, a Xenopus Rel/NF-kappa B-related gene, is expressed in the forebrain, dorsal aspect of the mid- and hindbrain, the otocysts and notochord of neurula and larval stage embryos. Overexpression of Xrel3 causes formation of embryonic tumours. We now show that Xrel3-induced tumours and animal caps from embryos injected with Xrel3 RNA express Otx2, Shh and Gli1. Heterodimerisation of a C-terminally deleted mutant of Xrel3 with wild-type Xrel3 inhibits in vitro binding of wild-type Xrel3 to Rel/NF-kappa B consensus DNA sequences. This dominant interference mutant disrupts Shh, Gli1 and Otx2 mRNA patterning and inhibits anterior development when expressed in the dorsal side of zygotes, which is rescued by co-injecting wild-type Xrel3 mRNA. In chick development, Rel activates Shh signalling, which is required for normal limb formation; Shh, Gli1 and Otx2 encode important neural patterning elements in vertebrates. The activation of these genes in tumours by Xrel3 overexpression and the inhibition of their expression and head development by a dominant interference mutant of Xrel3 indicates that Rel/NF-kappa B is required for activation of these genes and for anterior neural patterning in Xenopus.

Binding of myc proteins to canonical and noncanonical DNA sequences

1993 ◽  
Vol 13 (9) ◽  
pp. 5216-5224
Author(s):  
T K Blackwell ◽  
J Huang ◽  
A Ma ◽  
L Kretzner ◽  
F W Alt ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Site Selection ◽  
Wild Type ◽  
In Vitro Binding ◽  
Helix Loop Helix ◽  
Important Amino Acid ◽  
Vitro Binding ◽  
Binding Sequence ◽  
Half Site

Using an in vitro binding-site selection assay, we have demonstrated that c-Myc-Max complexes bind not only to canonical CACGTG or CATGTG motifs that are flanked by variable sequences but also to noncanonical sites that consist of an internal CG or TG dinucleotide in the context of particular variations in the CA--TG consensus. None of the selected sites contain an internal TA dinucleotide, suggesting that Myc proteins necessarily bind asymmetrically in the context of a CAT half-site. The noncanonical sites can all be bound by proteins of the Myc-Max family but not necessarily by the related CACGTG- and CATGTG-binding proteins USF and TFE3. Substitution of an arginine that is conserved in these proteins into MyoD (MyoD-R) changes its binding specificity so that it recognizes CACGTG instead of the MyoD cognate sequence (CAGCTG). However, like USF and TFE3, MyoD-R does not bind to all of the noncanonical c-Myc-Max sites. Although this R substitution changes the internal dinucleotide specificity of MyoD, it does not significantly alter its wild-type binding sequence preferences at positions outside of the CA--TG motif, suggesting that it does not dramatically change other important amino acid-DNA contacts; this observation has important implications for models of basic-helix-loop-helix protein-DNA binding.

scholarly journals Binding of myc proteins to canonical and noncanonical DNA sequences.

1993 ◽  
Vol 13 (9) ◽  
pp. 5216-5224 ◽  
Author(s):  
T K Blackwell ◽  
J Huang ◽  
A Ma ◽  
L Kretzner ◽  
F W Alt ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Site Selection ◽  
Wild Type ◽  
In Vitro Binding ◽  
Helix Loop Helix ◽  
Important Amino Acid ◽  
Vitro Binding ◽  
Binding Sequence ◽  
Half Site

Using an in vitro binding-site selection assay, we have demonstrated that c-Myc-Max complexes bind not only to canonical CACGTG or CATGTG motifs that are flanked by variable sequences but also to noncanonical sites that consist of an internal CG or TG dinucleotide in the context of particular variations in the CA--TG consensus. None of the selected sites contain an internal TA dinucleotide, suggesting that Myc proteins necessarily bind asymmetrically in the context of a CAT half-site. The noncanonical sites can all be bound by proteins of the Myc-Max family but not necessarily by the related CACGTG- and CATGTG-binding proteins USF and TFE3. Substitution of an arginine that is conserved in these proteins into MyoD (MyoD-R) changes its binding specificity so that it recognizes CACGTG instead of the MyoD cognate sequence (CAGCTG). However, like USF and TFE3, MyoD-R does not bind to all of the noncanonical c-Myc-Max sites. Although this R substitution changes the internal dinucleotide specificity of MyoD, it does not significantly alter its wild-type binding sequence preferences at positions outside of the CA--TG motif, suggesting that it does not dramatically change other important amino acid-DNA contacts; this observation has important implications for models of basic-helix-loop-helix protein-DNA binding.

scholarly journals Severe factor VII deficiency caused by mutations abolishing the cleavage site for activation and altering binding to tissue factor

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3524-3535 ◽  
Author(s):  
S Chaing ◽  
B Clarke ◽  
S Sridhara ◽  
K Chu ◽  
P Friedman ◽  
...  
Keyword(s):  
Amino Acid ◽  
Tissue Factor ◽  
Time Course ◽  
Binding Assay ◽  
Factor Vii ◽  
Wild Type ◽  
Vitro Binding ◽  
Epidermal Growth ◽  
A Site

Abstract Factor VII (F.VII) is a vitamin-K-dependent serine protease required in the early stages of blood coagulation. We describe here a patient with severe F.VII deficiency, with a normal plasma F.VII antigen level (452 ng/mL) and F.VII activity less than 1%, who is homozygous for two defects: a G-->A transition at nucleotide 6055 in exon 4, which results in an Arg-->Gln change at amino acid 79 (R79Q); and a G-->A transition at nucleotide 8961 in exon 6, which results in an Arg-->Gln substitution at amino acid 152 (R152Q). The R79Q mutation occurs in the first epidermal growth factor (EGF)-like domain, which has previously been implicated in binding to tissue factor. The R152Q mutation occurs at a site (Arg 152-Ile 153) that is normally cleaved to generate activated F.VII (F.VIIa). Analysis of purified F.VII from patient plasma shows that the material cannot be activated by F.Xa and cofactors. In addition, in an in vitro binding assay using relipidated recombinant tissue factor, patient plasma showed markedly reduced binding to tissue factor at all concentrations tested. In an effort to separate the contributions of the two mutations, three recombinant variants, wild-type, R79Q, and R152Q, were prepared and analyzed. The R152Q variant had markedly reduced activity in a clotting assay, whereas R79Q showed a milder, concentration-dependent reduction. The R152Q variant exhibited nearly normal binding in the tissue factor binding assay, whereas the R79Q variant had markedly reduced binding. The time course of activation of the R79Q variant was slowed compared with wild-type. Our results suggest that the first EGF-like domain is required for binding to tissue factor and that the F.VII zymogen lacks activity and requires activation for expression of biologic activity.

Organization of the noncontiguous promoter components of adenovirus VAI RNA gene is strikingly similar to that of eucaryotic tRNA genes

1983 ◽  
Vol 3 (11) ◽  
pp. 1996-2005
Author(s):  
R A Bhat ◽  
B Metz ◽  
B Thimmappaya
Keyword(s):  
Dna Sequences ◽  
Transcriptional Control ◽  
Transcriptional Activity ◽  
Evolutionary Relationship ◽  
Trna Genes ◽  
Wild Type ◽  
Base Pairs ◽  
Internal Promoter ◽  
Cell Extracts

The intragenic transcriptional control region (internal promoter) of the adenovirus type 2 VAI RNA gene was mutated by deletion, insertion, and substitution of DNA sequences at the plasmid level. The mutant plasmids were assayed for in vitro transcriptional activity by using HeLa cell extracts. The mutant clones with substitution or insertion of DNA sequences or both between nucleotides +18 and +53 of the VAI RNA gene were all transcriptionally active, although to various extents. Substitution of unrelated DNA sequences up to +26 or between +54 and +61 abolished the transcriptional activity completely. Based on these results, the intragenic promoter sequences of the VAI RNA gene can be subdivided into two components: element A, +10 to +18; and element B, +54 to +69. The distance between the A and B components could be enlarged from its normal 35 base pairs to 75 base pairs without destroying the transcriptional activity. However, a deletion of 4 or 6 base pairs in the DNA segment separating the A and B components (segment C) reduced the transcriptional activity of the genes to less than 2% of that of the wild type. When the VAI RNA gene with its element A or B was substituted for the corresponding element A or B of the Xenopus laevis tRNAMet gene, the hybrid genes transcribed close to the level of the wild-type VAI RNA gene and about 10- to 20-fold more efficiently than the tRNAMet gene. Thus, the organization of DNA sequences in the internal promoter of the VAI RNA gene appears to be very similar to that of eucaryotic tRNA genes. This similarity suggests an evolutionary relationship of the VAI RNA gene to tRNA genes.

scholarly journals Location of sequences in polyomavirus DNA that are required for early gene expression in vivo and in vitro.

1984 ◽  
Vol 4 (12) ◽  
pp. 2594-2609 ◽  
Author(s):  
C R Mueller ◽  
A M Mes-Masson ◽  
M Bouvier ◽  
J A Hassell
Keyword(s):  
Gene Expression ◽  
Thymidine Kinase ◽  
Dna Sequences ◽  
Viral Genome ◽  
Simian Virus 40 ◽  
Early Gene ◽  
Wild Type ◽  
Transcription In Vitro

To define the DNA sequences required for the expression of the polyomavirus early transcription unit, we cloned part of the viral genome in a plasmid vector, isolated mutants bearing lesions introduced in vitro within DNA sequences upstream of the transcriptional start site, and measured the capacity of these various mutant genomes to transform cells and to function as templates for transcription in vitro by comparison with wild-type DNA. One set of mutants bore 5' unidirectional deletions beginning at position -810 and extending downstream to position +4. Another set of mutants bore 3' undirectional deletions starting at position +4 and progressing upstream to position -311. The last set of mutants bore internal deletions between positions -810 and +4. Analyses of the properties of these mutant DNAs led us to conclude that the region between positions -403 and -311 includes an enhancer of gene expression. Deletion of this area from the viral genome reduced gene expression in vivo to 1 to 2% of wild-type levels, as measured by transformation assays. Moreover, this region increased the frequency of transformation of thymidine kinase-negative Rat-2 cells by the herpes simplex virus thymidine kinase (tk) gene from 5- to 20-fold. This occurred only if the polyomavirus sequences were covalently linked to the tk gene and then occurred independently of their orientation or position relative to the tk gene. A second transcriptional element is located downstream of the enhancer between positions -311 and -213. This element together with the enhancer was sufficient to bring about transformation of Rat-1 cells at nearly wild-type frequencies, and together these elements constitute the minimal sequences required for gene expression in vivo. The sequences making up the second element may be functionally duplicated downstream of position -165 (between positions -165 and -60). This was revealed by the characterization of mutant genomes with deletions between positions -349 and -60. The role of these redundant elements is not known; however, they may be analogous to the 21-base-pair repeats of simian virus 40. Finally, sequences between positions -57 and -1 were required for accurate and efficient transcription in vitro. However, this DNA stretch, which includes the TATA box and major transcriptional start sites, was not absolutely required for gene expression in vivo. We conclude that the polyomavirus promoter comprises multiple functional elements which are distributed across a DNA stretch of about 400 base pairs.

scholarly journals Meis proteins are major in vivo DNA binding partners for wild-type but not chimeric Pbx proteins.

1997 ◽  
Vol 17 (10) ◽  
pp. 5679-5687 ◽  
Author(s):  
C P Chang ◽  
Y Jacobs ◽  
T Nakamura ◽  
N A Jenkins ◽  
N G Copeland ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Cultured Cells ◽  
Binding Activity ◽  
Wild Type ◽  
Hox Proteins ◽  
Amino Terminal ◽  
Binding Partners

The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1.

scholarly journals Differences between MyoD DNA binding and activation site requirements revealed by functional random sequence selection.

1996 ◽  
Vol 16 (7) ◽  
pp. 3893-3900 ◽  
Author(s):  
J Huang ◽  
T K Blackwell ◽  
L Kedes ◽  
H Weintraub
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
Random Sequence ◽  
In Vitro Binding ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Vitro Binding

A method has been developed for selecting functional enhancer/promoter sites from random DNA sequences in higher eukaryotic cells. Of sequences that were thus selected for transcriptional activation by the muscle-specific basic helix-loop-helix protein MyoD, only a subset are similar to the preferred in vitro binding consensus, and in the same promoter context an optimal in vitro binding site was inactive. Other sequences with full transcriptional activity instead exhibit sequence preferences that, remarkably, are generally either identical or very similar to those found in naturally occurring muscle-specific promoters. This first systematic examination of the relation between DNA binding and transcriptional activation by basic helix-loop-helix proteins indicates that binding per se is necessary but not sufficient for transcriptional activation by MyoD and implies a requirement for other DNA sequence-dependent interactions or conformations at its binding site.

GTPase-activating protein and phosphatidylinositol 3-kinase bind to distinct regions of the platelet-derived growth factor receptor beta subunit

1992 ◽  
Vol 12 (6) ◽  
pp. 2534-2544
Author(s):  
A Kazlauskas ◽  
A Kashishian ◽  
J A Cooper ◽  
M Valius
Keyword(s):  
Beta Subunit ◽  
Platelet Derived Growth Factor ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Gtpase Activating Protein ◽  
Tyrosine Residues ◽  
In Vitro Binding ◽  
Pi3k Activity ◽  
Vitro Binding

In response to binding of platelet-derived growth factor (PDGF), the PDGF receptor (PDGFR) beta subunit is phosphorylated on tyrosine residues and associates with numerous signal transduction enzymes, including the GTPase-activating protein of ras (GAP) and phosphatidylinositol 3-kinase (PI3K). Previous studies have shown that association of PI3K requires phosphorylation of tyrosine 751 (Y751) in the kinase insert and that this region of receptor forms at least a portion of the binding site for PI3K. In this study, the in vitro binding of GAP to the PDGFR was investigated. Like PI3K, GAP associates only with receptors that have been permitted to autophosphorylate, and GAP itself does not require tyrosine phosphate in order to stably associate with the phosphorylated PDGFR. To define which tyrosine residues are required for GAP binding, a panel of PDGFR phosphorylation site mutants was tested. Mutation of Y771 reduced the amount of GAP that associates to an undetectable level. In contrast, the F771 (phenylalanine at 771) mutant bound wild-type levels of PI3K, whereas the F740 and F751 mutants bound 3 and 23%, respectively, of the wild-type levels of PI3K but wild-type levels of GAP. The F740/F751 double mutant associated with wild-type levels of GAP, but no detectable PI3K activity, while the F740/F751/F771 triple mutant could not bind either GAP or PI3K. The in vitro and in vivo associations of GAP and PI3K activity to these PDGFR mutants were indistinguishable. The distinct tyrosine residue requirements suggest that GAP and PI3K bind different regions of the PDGFR. This possibility was also supported by the observation that the antibody to the PDGFR kinase insert Y751 region that blocks association of PI3K had only a minor effect on the in vitro binding of GAP. In addition, highly purified PI3K and GAP associated in the absence of other cellular proteins and neither cooperated nor competed with each other's binding to the PDGFR. Taken together, these studies indicate that GAP and PI3K bind directly to the PDGFR and have discrete binding sites that include portions of the kinase insert domain.

scholarly journals A Naturally Occurring Deletion in FliE from Salmonella enterica Serovar Dublin Results in an Aflagellate Phenotype and Defective Proinflammatory Properties

2017 ◽  
Vol 86 (1) ◽  
Author(s):  
Sebastián Sasías ◽  
Adriana Martínez-Sanguiné ◽  
Laura Betancor ◽  
Arací Martínez ◽  
Bruno D'Alessandro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Sequences ◽  
High Frequency ◽  
Salmonella Enterica ◽  
Wild Type ◽  
Content Type ◽  
Naturally Occurring ◽  
Salmonella Enterica Serovar Dublin

ABSTRACTSalmonella entericaserovar Dublin is adapted to cattle but is able to infect humans with high invasiveness. An acute inflammatory response at the intestine helps to preventSalmonelladissemination to systemic sites. Flagella contribute to this response by providing motility and FliC-mediated signaling through pattern recognition receptors. In a previous work, we reported a high frequency (11 out of 25) ofS. Dublin isolates lacking flagella in a collection obtained from humans and cattle. The aflagellate strains were impaired in their proinflammatory propertiesin vitroandin vivo. The aim of this work was to elucidate the underlying cause of the absence of flagella inS. Dublin isolates. We report here that class 3 flagellar genes are repressed in the human aflagellate isolates, due to impaired secretion of FliA anti-sigma factor FlgM. This phenotype is due to an in-frame 42-nucleotide deletion in thefliEgene, which codes for a protein located in the flagellar basal body. The deletion is predicted to produce a protein lacking amino acids 18 to 31. The aflagellate phenotype was highly stable; revertants were obtained only whenfliAwas artificially overexpressed combined with several successive passages in motility agar. DNA sequence analysis revealed that motile revertants resulted from duplications of DNA sequences infliEadjacent to the deleted region. These duplications produced a FliE protein of similar length to the wild type and demonstrate that amino acids 18 to 31 of FliE are not essential. The same deletion was detected inS. Dublin isolates obtained from cattle, indicating that this mutation circulates in nature.

scholarly journals Chromosome-Based Genetic Complementation System for Xylella fastidiosa

2009 ◽  
Vol 75 (6) ◽  
pp. 1679-1687 ◽  
Author(s):  
Ayumi Matsumoto ◽  
Glenn M. Young ◽  
Michele M. Igo
Keyword(s):  
Dna Sequences ◽  
Xylella Fastidiosa ◽  
Cell Physiology ◽  
Multiple Cloning Site ◽  
Wild Type ◽  
In Planta ◽  
Catalase Peroxidase ◽  
The Stability

ABSTRACT Xylella fastidiosa is a xylem-limited, gram-negative bacterium that causes Pierce's disease of grapevine. Here, we describe the construction of four vectors that facilitate the insertion of genes into a neutral site (NS1) in the X. fastidiosa chromosome. These vectors carry a colE1-like (pMB1) replicon and DNA sequences from NS1 flanking a multiple-cloning site and a resistance marker for one of the following antibiotics: chloramphenicol, erythromycin, gentamicin, or kanamycin. In X. fastidiosa, vectors with colE1-like (pMB1) replicons have been found to result primarily in the recovery of double recombinants rather than single recombinants. Thus, the ease of obtaining double recombinants and the stability of the resulting insertions at NS1 in the absence of selective pressure are the major advantages of this system. Based on in vitro and in planta characterizations, strains carrying insertions within NS1 are indistinguishable from wild-type X. fastidiosa in terms of growth rate, biofilm formation, and pathogenicity. To illustrate the usefulness of this system for complementation analysis, we constructed a strain carrying a mutation in the X. fastidiosa cpeB gene, which is predicted to encode a catalase/peroxidase, and showed that the sensitivity of this mutant to hydrogen peroxide could be overcome by the introduction of a wild-type copy of cpeB at NS1. Thus, this chromosome-based complementation system provides a valuable genetic tool for investigating the role of specific genes in X. fastidiosa cell physiology and virulence.

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