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PubMed2000

Characterization of a maternal T-Box gene in Ciona intestinalis.

A new T-box gene, CiVegTR, was isolated in the ascidian Ciona intestinalis. CiVegTR maternal RNAs become localized to the vegetal cytoplasm of fertilized eggs and are incorporated into muscle lineages derived from the B4.1 blastomere. The CiVegTR protein binds to specific sequences within a minimal, 262-bp enhancer that mediates Ci-snail expression in the tail muscles. Mutations in these binding sites abolish expression from an otherwise normal lacZ reporter gene in electroporated embryos. In addition to the previously identified AC-core E-box sequences, T-box recognition sequences are conserved in the promoter regions of many genes expressed in B4.1 lineages in both Ciona and the distantly related ascidian Halocynthia. These results suggest that CiVegTR encodes a component of the classical muscle determinant that was first identified in ascidians nearly 100 years ago.

Amino Acid Sequence↗

PubMed2002

Identification of a unique family of F-box proteins in atadenoviruses.

Ovine adenovirus isolate 287 (OAdV-7) is the prototype of the atadenoviruses, a genus whose strategy for infection and replication is still being elucidated. A transcription unit at the right end of the genome contains four related genes (ORFs RH1, 2, 4, and 6), at least three of which are nonessential for replication in vitro. Related genes are also present in the genomes of bovine and avian atadenoviruses. To investigate how these apparently redundant genes are decoded, a more detailed transcription map of the right end of the OAdV-7 genome has been deduced. Eight transcripts that were derived from a promoter in the terminal repeat sequence were identified. Five were potentially bicistronic. The transcripts could encode all the potential proteins of the region subject to efficient reinitiation of translation. However, the most interesting and surprising finding in this work was that the related RH proteins carry an F-box motif. This was first identified in OAdV-7 RH1 and subsequently found in RH2, 4, and 6 proteins and the related reading frames from the bovine and avian atadenoviruses. Although very rare among viral proteins, several hundred cellular proteins contain F-box motifs. F-box proteins facilitate the degradation of a variety of important regulatory proteins via SCF ubiquitin ligase complexes. Thus, it appears that atadenoviruses have adopted a strategy to regulate a key cellular pathway(s) that distinguishes them from the other adenovirus genera and from most viruses in general.

Adenoviridae↗

PubMed1980

[Analysis of a boxing match with fatal outcome resulting from unusually severe brain damage (author's transl)].

A boxing match with fatal outcome is described. The boxer received 21 punches in the nape of his neck, as well as many other blows. After a knock out in the 15th round he conceded the fight; some minutes later he suddenly collapsed. Deep unconsciousness, maximal dilated pupils and extensor-spasms were observed but respiration continued. Although it was possible to evacuate a subdural hematoma 30 to 45 min later, a high-grade cerebral oedema remained resistant to therapy. In addition hypoxemia caused disseminated necroses of heart-muscle fibres. Intensive hemorrhages were detected in the cervical muscles and in the dorsal parts of the intervertebral discs on the level of C 2/3 and C 3/4. Neuropathological investigation revealed the typical alterations brought about by craniotomy carried out for a subdural hematoma caused by the tearing off of Vv. cerebralis superiores, as well as extensive contusion hemorrhages. The high-grade cerebral oedema caused congestion and dislocation which led to massive hemorrhages, hypoxemia to unusually extensive hemorrhagical and white necroses of cortex and brain stem ganglions. Special attention is paid to the pathogenesis of these findings. A report on boxing--particularly professional boxing--issued by the Federal Institute of Sport Medicine in Cologne in 1977 is critically analysed.

Adult↗

PubMed2003

Isolation and characterization of two T-box genes from sponges, the phylogenetically oldest metazoan taxon.

It is now well established that all metazoan phyla derived from one common ancestor, the hypothetical Urmetazoa. Due to the basal position of Porifera (Demospongiae) in the phylogenetic tree of Metazoa, studies on the mechanisms controlling the development of these animals can provide clues on the understanding of the origin of multicellular animals and on how the first organization of the body plan evolved. In this report we describe the isolation and genomic characterization of two T-box genes from the siliceous sponge Suberites domuncula. The phylogenetic analysis classifies one into the subfamily of Brachyury, Sd-Bra, and the second into the Tbx2 subfamily, Sd-Tbx2. Analyses of the Sd-Bra and Sd-Tbx2 sequences and their intron-exon structures demonstrate their basal position in the phylogeny of the T-box family, and allows us to hypothesize a model of the phylogenetic evolution of all T-box genes. Furthermore, we report the presence of two different products of alternative splicing of Sd-Bra, and demonstrate that they exist in different phosphorylation and glycosylation states in the sponge tissue. Sd-Bra expression in tissue and 3D-cell aggregates (primmorphs) is analyzed, suggesting that Sd-Bra might also have a role in Porifera morphogenesis.

Alternative Splicing↗

PubMed2000

The evolution of paired appendages in vertebrates: T-box genes in the zebrafish.

The presence of two sets of paired appendages is one of the defining features of jawed vertebrates. We are interested in identifying genetic systems that could have been responsible for the origin of the first set of such appendages, for their subsequent duplication at a different axial level, and/or for the generation of their distinct identities. It has been hypothesized that four genes of the T-box gene family (Tbx2-Tbx5) played important roles in the course of vertebrate limb evolution. To test this idea, we characterized the orthologs of tetrapod limb-expressed T-box genes from a teleost, Danio rerio. Here we report isolation of three of these genes, tbx2, tbx4, and tbx5. We found that their expression patterns are remarkably similar to those of their tetrapod counterparts. In particular, expression of tbx5 and tbx4 is restricted to pectoral and pelvic fin buds, respectively, while tbx2 can be detected at the anterior and posterior margins of the outgrowing fin buds. This, in combination with conserved expression patterns in other tissues, suggests that the last common ancestor of teleosts and tetrapods possessed all four of these limb-expressed T-box genes (Tbx2-Tbx5), and that these genes had already acquired, and have subsequently maintained, their gene-specific functions. Furthermore, this evidence provides molecular support for the notion that teleost pectoral and pelvic fins and tetrapod fore- and hindlimbs, respectively, are homologous structures, as suggested by comparative morphological analyses.

Amino Acid Sequence↗

PubMed1992

Parvovirus H-1 P38 promoter requires the trans-activation region (tar), an SP1 site, and a TATA box for full activity.

In the parvovirus H-1 P38 promoter, there are sequences identified as a TATA box, an SP1 site, and a trans-activation responsive element (tar). It was previously shown that the parvovirus H-1 nonstructural protein NS1 positively regulates the expression of the P38 promoter for the viral capsid protein gene via the tar. To characterize the tar element further, a series of single-point mutations of the tar was constructed and the mutants were compared to wild-type for the trans-activation of the P38 promoter using a cat reporter gene. Most of the tar mutations had a negative effect on the P38 promoter and some of them reduced activity as much as 70%. However, when several mutants with multiple-point mutations in the tar were tested, no significant additive effect was observed. We examined the function of the SP1 site in the trans-activation of the P38 promoter by replacing the wild-type SP1 sequence with synthetic DNA fragments, OSP1 or 2SP1, containing no SP1 or two SP1 sites respectively, in a P38 construct with a cat reporter gene. The results indicate that P38 expression varies in proportion to the number of SP1 sites, suggesting a role for the SP1 site during trans-activation by NS1. The role of the TATA box on the P38 promoter was also examined by mutagenizing TATA to CACG. The activity of this promoter was reduced to 43%. When a construct mutated at both the SP1 and TATA box sites was tested for its activity, about 22% of the wild-type activity remained, implying that this remaining activity was contributed largely by the tar element. A model is proposed for how the tar element activates the wild-type and SP1-TATA minus promoters in the presence of NS1.

Base Sequence↗

PubMed1992

A synthetic early promoter from a baculovirus: roles of the TATA box and conserved start site CAGT sequence in basal levels of transcription.

Many baculovirus early genes and insect genes transcribed by RNA polymerase II have a conserved transcription start site sequence (CAGT) located downstream of a consensus TATA box. To examine the functions and interactions of these two motifs in initiating accurately positioned basal transcription, a 43-nt synthetic promoter was synthesized from the TATA box and start site sequences of the gp64 early promoter from the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). The synthetic promoter initiated accurately and was also transactivated by the baculovirus transcriptional activator, IE1. To determine the roles of sequences within the 43-nt synthetic promoter, a series of linker-scanning and spacing mutations were analyzed for transcriptional activity, start site selection, and transactivation. Linker-scanning mutations were examined in vivo by transient expression and reporter gene assays. To examine transcription start site selection, promoter constructs were used for in vitro transcription in nuclear extracts from uninfected Spodoptera frugiperda (Sf9) cells. In vivo and in vitro analyses show that the TATA box, and not the start site CAGT, is the primary element controlling start site selection. Substitution of the conserved start site CAGT sequence resulted in a reduction of both reporter gene activity and in vitro transcripts, although transcripts initiated accurately. Data from linker-scanning and spacing mutations indicate that the conserved start site CAGT sequences are not required for accurate initiation but sequences at the start site play an important role in initiation efficiency.

Baculoviridae↗

PubMed1996

A Drosophila melanogaster homologue of the human DEAD-box gene DDX1.

DEAD-box genes are found throughout evolution and encode RNA-binding proteins. Such proteins include eukaryotic initiation factor-4A, which is essential for protein translation, Vasa, which is essential for germ line development, and a number of nuclear and mitochondrial RNA splicing factors. Transcription of a human DEAD-box gene, DDX1, is elevated in two retinoblastoma cell lines as a result of amplification of the immediate chromosomal region surrounding it, suggesting an important role for this gene in control of cell growth and division. We have isolated a Drosophila melanogaster (Dm) homologue (Ddx1) of DDX1 which is strikingly similar to the human gene. The similarity (58.3% amino acid (aa) identity over 720 aa) extends beyond regions conserved in all DEAD-box proteins and covers the entire lengths of the proteins. The 2.7-kb Dm Ddx1 RNA is expressed throughout development, but its levels are elevated in early embryos. Ddx1 maps to polytene chromosome band 79D4 on the left arm of Dm chromosome 3.

Amino Acid Sequence↗

PubMed2005

Characterization of FBX25, encoding a novel brain-expressed F-box protein.

F-box proteins (FBPs) confer substrate specificity to the SCF-type (Skp1/Cul1/FBP) of ubiquitin ligase complexes through their F-box. Multiple FBPs have been predicted, but experimental evidence is lagging. We report on the predicted human FBP hFBX25 which we found to be disrupted in a mentally retarded translocation carrier suffering from epileptic seizures. We investigated hFBX25's genomic organization and established hFBX25 as an FBP by verifying its interaction with Skp1 and Cul1. In the process, we identified an atypical serine residue in the F-box which is crucial for the hFBX25-Skp1 binding. We determined hFBX25's subcellular localization. We found strong transcription in human brain. In mouse embryonic sections, mFbx25 shows predominantly neuronal expression and in adult mouse brain, expression is confined to the hippocampus, the cerebral cortex and the Purkinje cell layer. Interestingly, aberrations in the ubiquitin pathway have been linked to neurological conditions.

Amino Acid Sequence↗

PubMed2004

Targeted degradation of beta-catenin by chimeric F-box fusion proteins.

Adenomatous polyposis coli (APC) tumor suppressor protein, together with Axin and glycogen synthase kinase 3beta (GSK-3beta), forms a Wnt-regulated signaling complex that mediates phosphorylation-dependent degradation of cytoplasmic beta-catenin by ubiquitin-dependent proteolysis. Degradation of phosphorylated beta-catenin is initiated by interaction through the WD40-repeat of a F-box protein beta-TrCP, a component of SCF ubiquitin ligase complex. Mutations in APC, Axin, and beta-catenin that prevent down-regulation of cytoplasmic beta-catenin are found in various types of cancers. In the search for efficient treatment and prevention of malignancies associated with increased levels of cytoplasmic beta-catenin, we created chimeric F-box fusion proteins by replacing the WD40-repeat of beta-TrCP with the beta-catenin-binding domains of Tcf4 and E-cadherin. Expression of chimeric F-box fusion proteins successfully promotes degradation of beta-catenin independently of GSK-3beta-mediated phosphorylation. More importantly, this degradation does not require intact APC protein (pAPC).

Adenomatous Polyposis Coli Protein↗

PubMed2006

The presence of a common downstream box enables the simultaneous expression of multiple proteins in an E. coli extract.

In our experiments to produce different combinations of recombinant proteins in a cell-free protein synthesis system derived from Escherichia coli, we found that certain pairs of ORFs were not expressed equally. Instead, only a single DNA species was expressed dominantly, while the expression of the others was almost completely repressed. This bias during the co-expression of the DNA pairs was eliminated when an identical downstream box sequence was added to the 5'-ends of the template DNA pairs. By introducing identical nucleotide sequences of the his-tag or the downstream box of chloramphenicol acetyltransferase (CAT-DB) in front of the target genes that were otherwise not expressed compatibly, both of the encoded proteins were produced at similar productivities. Moreover, in the presence of a common downstream box, multiple genes were simultaneously expressed in the same reaction mixture. We expect that the proposed approach will offer a powerful tool for the preparation of unbiased protein libraries, as well as for studying the structure and functions of interacting proteins.

Cell Extracts↗

PubMed2006

T-box proteins differentially activate the expression of the endogenous interferon gamma gene versus transfected reporter genes in non-immune cells.

The T-box transcription factor T-bet is expressed in a number of hematopoetic cell types and plays an essential role in the lineage determination of Th1 T-helper cells. In the absence of T-bet, CD4(+) T-cells fail to induce IFNgamma, the cytokine whose expression characterizes Th1 cells. Here we show that, surprisingly, T-bet induces the expression of endogenous IFNgamma in non-immune human cells, including 293 and other cell lines. Thus T-bet can induce IFNgamma expression independently of its role in T-cell lineage determination. In addition, mutations in T-bet, and chimeras of T-bet with other transcription factors including the T-box transcription factor, TBX2, differentially affect the ability of T-bet to activate expression of endogenous IFNgamma versus a T-site regulated reporter gene. A truncated T-betVp16 fusion protein strongly activates the T-site reporter but fails to activate endogenous IFNgamma. Conversely, native T-bet strongly activates endogenous IFNgamma expression but only weakly activates the reporter gene. Fusion of the Vp16 activation domain to full-length T-bet greatly increases its activation of both endogenous IFNgamma and transfected T-site reporter gene expression. In contrast, TBX2Vp16 potently activates the T-site reporter but has a negligible effect on endogenous IFNgamma expression. Butyrate treatment of T-bet expressing cells potentiates the expression of endogenous IFNgamma but weakly represses expression of the T-site reporter gene. These data indicate that induction of endogenous IFNgamma can be uncoupled from differentiation into the Th1 lineage and that the expression of endogenous IFNgamma versus a T-site reporter gene is differentially regulated by T-bet and other T-box proteins.

Base Sequence↗

PubMed2006

SRY and human sex determination: the basic tail of the HMG box functions as a kinetic clamp to augment DNA bending.

Human testis-determining factor SRY contains a high-mobility-group (HMG) box, an alpha-helical DNA-binding domain that binds within an expanded minor groove to induce DNA bending. This motif is flanked on the C-terminal end by a basic tail, which functions both as a nuclear localization signal and accessory DNA-binding element. Whereas the HMG box is broadly conserved among otherwise unrelated transcription factors, tails differ in sequence and mode of DNA binding. Contrasting examples are provided by SRY and lymphoid enhancer factor 1 (LEF-1): whereas the SRY tail remains in the minor groove distal to the HMG box, the LEF-1 tail binds back across the center of the bent DNA site. The LEF-1 tail relieves electrostatic repulsion that would otherwise be incurred within the compressed major groove to enable sharp DNA bending with high affinity. Here, we demonstrate that the analogous SRY tail functions as a "kinetic clamp" to regulate the lifetime of the bent DNA complex. As in LEF-1, partial truncation of the distal SRY tail reduces specific DNA affinity and DNA bending, but these perturbations are modest: binding is reduced by only 1.8-fold, and bending by only 7-10 degrees . "Tailed" and truncated SRY complexes exhibit similar structures (as probed by NMR) and distributions of long-range conformational substates (as probed by time-resolved fluorescence resonance energy transfer). Surprisingly, however, the SRY tail retards dissociation of the protein-DNA complex by 20-fold. The marked and compensating changes in rates of association and dissociation observed on tail truncation, disproportionate to perturbations in affinity or structure, suggest that this accessory element functions as a kinetic clamp to regulate the lifetime of the SRY-DNA complex. We speculate that the kinetic stability of a bent DNA complex is critical to the assembly and maintenance of a sex-specific transcriptional pre-initiation complex.

Amino Acid Sequence↗

PubMed2006

Involvement of DEAD-box proteins in group I and group II intron splicing. Biochemical characterization of Mss116p, ATP hydrolysis-dependent and -independent mechanisms, and general RNA chaperone activity.

The RNA-catalyzed splicing of group I and group II introns is facilitated by proteins that stabilize the active RNA structure or act as RNA chaperones to disrupt stable inactive structures that are kinetic traps in RNA folding. In Neurospora crassa and Saccharomyces cerevisiae, the latter function is fulfilled by specific DEAD-box proteins, denoted CYT-19 and Mss116p, respectively. Previous studies showed that purified CYT-19 stimulates the in vitro splicing of structurally diverse group I and group II introns, and uses the energy of ATP binding or hydrolysis to resolve kinetic traps. Here, we purified Mss116p and show that it has RNA-dependent ATPase activity, unwinds RNA duplexes in a non-polar fashion, and promotes ATP-independent strand-annealing. Further, we show that Mss116p binds RNA non-specifically and promotes in vitro splicing of both group I and group II intron RNAs, as well as RNA cleavage by the aI5gamma-derived D135 ribozyme. However, Mss116p also has ATP hydrolysis-independent effects on some of these reactions, which are not shared by CYT-19 and may reflect differences in its RNA-binding properties. We also show that a non-mitochondrial DEAD-box protein, yeast Ded1p, can function almost as efficiently as CYT-19 and Mss116p in splicing the yeast aI5gamma group II intron and less efficiently in splicing the bI1 group II intron. Together, our results show that Mss116p, like CYT-19, can act broadly as an RNA chaperone to stimulate the splicing of diverse group I and group II introns, and that Ded1p also has an RNA chaperone activity that can be assayed by its effect on splicing mitochondrial introns. Nevertheless, these DEAD-box protein RNA chaperones are not completely interchangeable and appear to function in somewhat different ways, using biochemical activities that have likely been tuned by coevolution to function optimally on specific RNA substrates.

Adenosine Triphosphate↗

PubMed2004

Precise nucleosome positioning and the TATA box dictate requirements for the histone H4 tail and the bromodomain factor Bdf1.

Acetylation of histone tails plays a key role in chromatin dynamics and is associated with the potential for gene expression. We show here that a 2-3 bp mispositioning of the nucleosome covering the TATA box at PHO5 induces a dependency on the acetylatable lysine residues of the histone H4 N-terminal region and on the TFIID-associated bromodomain factor Bdf1. This dependency arises either through fusion of the PHO5 promoter to a lacZ reporter or by mutation of the TATA box in the natural gene. The results suggest that promoters in which the TATA box is either absent or poorly accessible on the surface of a nucleosome may compensate by using Bdf1 bromodomains and acetylated H4 tails to anchor TFIID to the promoter during the initial stages of transcription activation. We propose that nucleosome positioning at the nucleotide level provides a subtle, but highly effective, mechanism for gene regulation.

Acetylation↗

PubMed2004

T-box binding sites are required for activity of a cardiac GATA-4 enhancer.

GATA-4 is a key regulator of a poorly understood cardiac morphogenetic program. We used genomic regions of the GATA-4 gene to target GFP expression to the developing heart of living fish. In these fish, GFP-expressing cells in the lateral plate mesoderm form two tubes that migrate ventrally to fuse into a linear heart tube. In addition, we find that a 14.8-kb fragment upstream of the transcription initiation site targets expression to both chambers and the valves of the heart. Truncation of 7 kb of the distal sequences eliminates expression in the atrium and the atrioventricular valve while expression is retained in the ventricle and the bulboventricular valve. Within this 7-kb distal regulatory region, we delineated a 1300-bp region with a cluster of consensus binding sites for T-box transcription factors. Mutation of these sequences significantly reduces reporter gene expression in the heart. This provides the first evidence that T-box factors function by directly regulating GATA-4 expression. Thus, GATA-4 regulatory elements control gene expression differentially along the rostro-caudal axis, and T-box binding elements in the GATA-4 promoter contribute to heart-specific expression.

Animals↗

PubMed2006

The T-box factor TBX-2 and the SUMO conjugating enzyme UBC-9 are required for ABa-derived pharyngeal muscle in C. elegans.

The C. elegans pharynx is produced from the embryonic blastomeres ABa and MS. Pharyngeal fate in the ABa lineage is specified by the combined activities of GLP-1/Notch-mediated signals and the TBX-37 and TBX-38 T-box transcription factors. Here, we show another T-box factor TBX-2 also functions in ABa-derived pharyngeal development. tbx-2 mutants arrest as L1 larvae lacking most or all ABa-derived pharyngeal muscles. In comparison, tbx-2 mutants retain ABa-derived marginal cells and pharyngeal muscles derived from MS. A tbx-2Colon, two colonsgfp translational fusion is expressed in a dynamic pattern in C. elegans embryos beginning near the 100-cell stage. Early expression is limited to a small number of cells, which likely include the ABa-derived pharyngeal precursors, while later expression is observed in body wall muscles and a subset of pharyngeal neurons. TBX-2 contains 2 consensus sumoylation sites, and it interacts in a yeast two-hybrid assay with the UBC-9 and GEI-17 components of the C. elegans SUMO-conjugating pathway. ubc-9(RNAi) has been previously shown to cause variable embryonic and larval arrest, and we find that, like tbx-2 mutants, ubc-9(RNAi) animals lack ABa-derived pharyngeal muscles. ubc-9(RNAi) also alters the subnuclear distribution of TBX-2::GFP fusion protein, suggesting that UBC-9 and TBX-2 interact in C. elegans. Together, these results indicate that TBX-2 and SUMO-conjugating enzymes are necessary for ABa-derived pharyngeal muscle, and we hypothesize that TBX-2 function requires sumoylation. Sumoylation is increasingly recognized as an important mechanism controlling activity of many nuclear factors, and these results provide the first evidence that T-box factor activity may require sumoylation.

Animals↗

PubMed2005

Phenylephrine requires the TATA box to activate transcription of GLUT1 in neonatal rat cardiac myocytes.

Cardiac hypertrophy and heart failure occur in association to alterations in glucose uptake and metabolism. Phenylephrine, among other hypertrophic agonists, has been reported to increase expression of GLUT1 in neonatal rat cardiac myocytes by activating transcription. However, the specific cis- or trans-acting factors in the GLUT1 gene that are targeted by this agonist remain elusive. Here we describe that the activity of the -99/+134 basal promoter of rat GLUT1 is increased by phenylephrine. Nevertheless, this is not mediated by previously described binding sites (GC-box, MG1E) in the promoter. Rather, the TATA box is required by the agonist to activate transcription from the promoter. Interestingly, The Ras-ERK mitogen-activated protein (MAP) kinase pathway is involved in the actions of phenylephrine on GLUT1 transcription, and the effects of Ras on the activity of the promoter depend on the integrity of the TATA box. Our data indicate that phenylephrine induces the expression of the TBP-associated factor TAF(II)250 mRNA, which increases in parallel to the expression of GLUT1, suggesting that altering the expression of basal transcription factors could be one mechanism by which phenylephrine may regulate the activity of the GLUT1 promoter.

Animals↗