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PubMed1994

Sequence determinants of the capping box, a stabilizing motif at the N-termini of alpha-helices.

The capping box, a recurrent hydrogen bonded motif at the N-termini of alpha-helices, caps 2 of the initial 4 backbone amide hydrogen donors of the helix (Harper ET, Rose GD, 1993, Biochemistry 32:7605-7609). In detail, the side chain of the first helical residue forms a hydrogen bond with the backbone of the fourth helical residue and, reciprocally, the side chain of the fourth residue forms a hydrogen bond with the backbone of the first residue. We now enlarge the earlier definition of this motif to include an accompanying hydrophobic interaction between residues that bracket the capping box sequence on either side. The expanded box motif--in which 2 hydrogen bonds and a hydrophobic interaction are localized within 6 consecutive residues--resembles a glycine-based capping motif found at helix C-termini (Aurora R, Srinivasan R, Rose GD, 1994, Science 264:1126-1130).

Amino Acid Sequence↗

PubMed1994

The yeast plasma membrane uracil permease is stabilized against stress induced degradation by a point mutation in a cyclin-like "destruction box".

Yeast uracil permease appears to be fairly stable in exponentially growing cells, but it undergoes rapid endocytosis followed by degradation when cells are submitted to adverse conditions, such as nutrient starvation or inhibition of protein synthesis. Uracil permease has a sequence (RIALGSLTD) that is very similar to the "destruction box" of mitotic cyclins. This box is required for the ubiquitin-dependent proteolysis of cyclins. We replaced the invariant arginine residue of the putative "destruction box" in uracil permease by an alanine. The mutation significantly protected the permease against stress-induced degradation. This result suggests that ligation to ubiquitin could be a signal for uracil permease degradation.

Amino Acid Sequence↗

PubMed1996

Crosslinking of SRF to the c-fos SRE CArG box guanines using photo-active thioguanine oligodeoxynucleotides.

6-Thioguanine (thioG) was chemically incorporated into 25-base oligodeoxynucleotides encoding the c-fos serum response element (SRE) at positions corresponding to each guanine of the CArG box, which only slightly impaired DNA binding by the Serum Response Factor (SRF). Upon exposure to long wavelength UV light each thioG-containing SRE could be crosslinked to SRF, with efficiencies ranging from < 1 to 25% of the complex depending on the position of thioG in the SRE and on the UV source used. Crosslinking was strongest to the 3' side of the CArG box, and to the outer rather than the inner CArG box guanines, consistent with hydrogen bonds formed between SRF and the outer guanines in the crystal structure [Pellegrini et al., Nature 376, 490, 1995]. The crosslinked product was found to be chemically unstable. Possible mechanisms of crosslink formation are discussed.

Cross-Linking Reagents↗

PubMed1997

Role of E boxes in the repression of E-cadherin expression.

Decreased expression of the intercellular adhesion molecule E-cadherin correlates with tumor aggressiveness and invasion capacity of cell lines. The decrease of E-cadherin expression results primarily from reduced mRNA expression. We show here that the activity of a human E-cadherin promoter construct is cell specific and correlates with E-cadherin mRNA expression. This spectrum of activity is conserved by a region as short as 81 bp obtained after 5' deletion. Mutation analysis revealed that two E box elements of this minimal promoter are involved in the silencing of E-cadherin promoter activity occurring in cancer cells. E boxes are mainly known as target sequences for bHLH transcription factors that are involved in the control of tissue differentiation and are antagonised by HLH proteins. However, mutation data and cotransfection experiments using HLH protein expression vectors indicate that bHLH transcription factors are not significantly involved in the E box mediated silencing of this E-cadherin promoter fragment.

Base Sequence↗

PubMed2000

A conserved role for H15-related T-box transcription factors in zebrafish and Drosophila heart formation.

T-box transcription factors are critical regulators of early embryonic development. We have characterized a novel zebrafish T-box transcription factor, hrT (H15-related T box) that is a close relative of Drosophila H15 and a recently identified human gene. We show that Drosophila H15 and zebrafish hrT are both expressed early during heart formation, in strong support of previous work postulating that vertebrate and arthropod hearts are homologous structures with conserved regulatory mechanisms. The timing and regulation of zebrafish hrT expression in anterior lateral plate mesoderm suggest a very early role for hrT in the differentiation of the cardiac precursors. hrT is coexpressed with gata4 and nkx2.5 not only in anterior lateral plate mesoderm but also in noncardiac mesoderm adjacent to the tail bud, suggesting that a conserved regulatory pathway links expression of these three genes in cardiac and noncardiac tissues. Finally, we analyzed hrT expression in pandora mutant embryos, since these have defects in many of the tissues that express hrT, including the heart. hrT expression is much reduced in the early heart fields of pandora mutants, whereas it is ectopically expressed subsequently. Using hrT expression as a marker, we describe a midline patterning defect in pandora affecting the anterior hindbrain and associated midline mesendodermal derivatives. We discuss the possibility that the cardiac ventricular defect previously described in pandora and the midline defects described here are related.

Amino Acid Sequence↗

PubMed1997

Newly identified paralogous groups on mouse chromosomes 5 and 11 reveal the age of a T-box cluster duplication.

A novel family of ancient transcription factors, the T-box family, involved in embryonic development in metazoans, was described recently. Four members of this family are grouped in two tightly linked pairs within the mouse genome. This arrangement can be explained by an original cluster formation followed by an en masse duplication. Here we demonstrate that this duplication event also included several closely linked genes. Using data obtained from linked paralogous genes, we show that the T-box cluster duplication occurred prior to the divergence between bony fish and tetrapods around 400 million years ago. This work facilitates our understanding of the status of the T-box gene family in different vertebrate lineages and also defines a novel paralogy group within the mouse genome.

Animals↗

PubMed1999

Hera from Thermus thermophilus: the first thermostable DEAD-box helicase with an RNase P protein motif.

DEAD-box proteins have been implicated in a wide array of cellular processes ranging from initiation of protein synthesis and ribosome biogenesis to mRNA splicing. Here, we report the isolation, biochemical characterization and crystallization of the first thermophilic DEAD box protein, Hera (heat-resistant RNA-dependent ATPase) from Thermus thermophilus HB8. The molecular mass of the deduced Hera protein sequence (510 amino acid residues) is 55.95 kDa. Hera possesses all of the conserved motifs found among the, DEAD-box RNA helicases. In addition, it also has a motif characteristic of the protein component of ribonuclease P at its C-terminal region (residues 372-386). Hera appears to be non-specific with respect to the RNA species that triggers ATPase activity. Nevertheless, at high temperature, ATPase activity is at a maximum when bacterial 16 S rRNA or 23 S rRNA are used as the substrates. Moreover, a deletion of the RNase P protein motif significantly reduces the ability of Hera to hydrolyze ATP in the presence of RNase P RNA. Hera has a specific ATPase activity of 480 units/microg and therefore, displays the highest ATPase specific activity reported for a protein of the RNA helicase family. We determined that Hera shows helix-destabilizing activity, and that the RNA-unwinding or helix-destabilizing activity of Hera is coupled to ATP hydrolysis. Since Hera is a stable thermophilic protein and we have obtained crystals of it diffracting beyond 2.6 A, the possibilities for structure determination of a full-length RNA-helicase are open.

Adenosine Triphosphatases↗

PubMed2000

Structural comparison of the PhoB and OmpR DNA-binding/transactivation domains and the arrangement of PhoB molecules on the phosphate box.

PhoB is a transcriptional activator that binds to the phosphate box in the promoters of the phosphate genes of Escherichia coli. PhoB contains two functional domains, an N-terminal phosphorylation domain and a C-terminal DNA-binding/transactivation domain. Here, the three-dimensional structure of the DNA-binding/transactivation domain has been determined by NMR. It consists of an N-terminal four-stranded beta-sheet, a central three helical bundle and a C-terminal beta-hairpin. The second and third helices form a helix-turn-helix (HTH) variant containing a longer turn than the corresponding turn of the classical HTH motif. The overall architecture is very close to that of the OmpR DNA-binding/transactivation domain, however, the conformation of the long turn region of PhoB, a putative interaction site for the RNA polymerase sigma subunit, is entirely different from that of the corresponding turn of OmpR, which interacts with the alpha subunit. In addition, the third helix of PhoB is three amino acid residues longer than the corresponding helix of OmpR. The binding site of PhoB is a TGTCA sequence and the phospahte box contains the two binding sites. NMR studies of the complexes of the PhoB DNA-binding/transactivation domain bound to several different DNA molecules have revealed that two PhoB molecules bind in a tandem array on the phosphate box. In each complex of PhoB the third helix of the DNA-binding/transactivation domain is likely to recognize the TGTCA sequence from the major groove of DNA and the C-terminal beta-hairpin contacts on the minor groove of the 3' site out of the TGTCA sequence in a non-specific manner. The long turn region facing outward is likely to interact with the sigma subunit.

Amino Acid Sequence↗

PubMed2000

Reconstitution of the KRAB-KAP-1 repressor complex: a model system for defining the molecular anatomy of RING-B box-coiled-coil domain-mediated protein-protein interactions.

The KRAB domain is a 75 amino acid residue transcriptional repression module commonly found in eukaryotic zinc-finger proteins. KRAB-mediated gene silencing requires binding to the corepressor KAP-1. The KRAB:KAP-1 interaction requires the RING-B box-coiled coil (RBCC) domain of KAP-1, which is a widely distributed motif, hypothesized to be a protein-protein interface. Little is known about RBCC-mediated ligand binding and the role of the individual sub-domains in recognition and specificity. We have addressed these issues by reconstituting and characterizing the KRAB:KAP-1-RBCC interaction using purified components. Our results show that KRAB binding to KAP-1 is direct and specific, as the related RBCC domains from TIF1alpha and MID1 do not bind the KRAB domain. A combination of gel filtration, analytical ultracentrifugation, chemical cross-linking, non-denaturing gel electrophoresis, and site-directed mutagenesis techniques has revealed that the KAP-1-RBCC must oligomerize likely as a homo-trimer in order to bind the KRAB domain. The RING finger, B2 box, and coiled-coil region are required for oligomerization of KAP-1-RBCC and KRAB binding, as mutations in these domains concomitantly abolished these functions. KRAB domain binding stabilized the homo-oligomeric state of the KAP-1-RBCC as detected by chemical cross-linking and velocity sedimentation studies. Mutant KAP-1-RBCC molecules hetero-oligomerize with the wild-type KAP-1, but these complexes were inactive for KRAB binding, suggesting a potential dominant negative activity. Substitution of the coiled-coil region with heterologous dimerization, trimerization, or tetramerization domains failed to recapitulate KRAB domain binding. Chimeric KAP-1-RBCC proteins containing either the RING, RING-B box, or coiled-coil regions from MID1 also failed to bind the KRAB domain. The KAP-1-RBCC mediates a highly specific, direct interaction with the KRAB domain, and it appears to function as an integrated, possibly cooperative structural unit wherein each sub-domain contributes to oligomerization and/or ligand recognition. These observations provide the first principles for RBCC domain-mediated protein-protein interaction and have implications for identifying new ligands for RBCC domain proteins.

Amino Acid Motifs↗

PubMed1998

HMG box containing transcription factors in lymphocyte differentiation.

The identification of the mammalian sex-determining gene Sry has led to the discovery of a large family of related ('HMG box') transcription factors that control developmental events in yeast, C. elegans, Drosophila and vertebrates. In lymphocyte differentiation, several HMG box proteins play a decisive role. Sox-4 is important for very early B-cell differentiation, while TCF-1/LEF-1 play a crucial role in early thymocyte development. TCF/LEF proteins have recently been found to constitute a downstream component of the Wingless/Wnt signal transduction pathway. In flies, this pathway controls segment polarity; in Xenopus it controls the definition of the body axis. Deregulation of the pathway occurs in several human tumors. These insights in the molecular events that are involved in TCF/LEF function in these organisms may eventually lead to the understanding of the function of these HMG box proteins in lymphoid development.

Animals↗

PubMed1994

Cloning of Rous sarcoma virus enhancer factor genes. I. Evidence that RSV-EF-I is related to Y-box (inverted CCAAT) binding proteins and binds to multiple motifs in the RSV enhancer.

A cDNA clone was isolated from a chicken embryo cDNA library employing a PCR-generated radiolabeled probe specific for the U3 region of the Rous sarcoma virus LTR. The cDNA encodes a protein of 345 aa and is homologous to the Y-box (inverted CCAAT) binding proteins. The amino acid sequence of the RSV-EF-I shows 75% identity with rat EF1 but the NH2-terminal 60-aa residues share little homology. At the carboxyl terminus an additional 28-aa sequence, rich in basic residues, probably encoded by an extra exon, is present in the chicken RSV-EF-1. Electrophoretic mobility assays carried out with various radiolabeled oligonucleotides spanning the U3 region of the RSV LTR (-234 to -54) indicated that the RSV-EF-I binds strongly to the sequence AAGGTGGTAC and somewhat less efficiently to the sequences AAGGAAAG and CTTATGCAA. In contrast to rat EF1A which binds to the inverted CCAAT box, RSV-EF-I does not bind to the CCAAT box sequence. These results suggest that the RSV-EF-I, although structurally similar to the rat EF1A, binds differently to more than one cis-acting element. The gene for RSV-EF-I is expressed in a variety of cell lines, although most abundantly in avian fibroblasts compared to mammalian cells. It is barely expressed in normal lives but expressed at significantly enhanced levels in many immortalized hepatocytes and hepatic tumors.

Amino Acid Sequence↗

PubMed1996

A novel MADS-box gene of potato (Solanum tuberosum L.) expressed during the early stages of tuberization.

A potato MADS-box gene cDNA (POTM1-1) from an early tuber cDNA library has been isolated and characterized. The deduced amino acid sequence of POTM1-1 cDNA encodes 250 amino acids of a putative transcription factor containing a MADS-box domain and a K-box domain. These conserved domains share high homologies to those of flower-specific homeotic proteins, TM4 of tomato and AP1 of Arabidopsis, indicating that POTM1-1 gene is a homologue of the AP1 gene family. The levels of POTM1-1 transcripts were high in axillary buds, underground stolen tips, and newly formed tubers, but relatively low in mature tubers. During axillary bud development in a model petiole-leaf cutting system, the levels of POTM1-1 transcripts were abundant in actively growing shoots and during the early stages of microtuber development. It is possible that POTM1-1 functions as a transcription factor that regulates plant developmental processes in a number of tissue types.

Base Sequence↗

PubMed1992

Functional elements of the Arabidopsis Adh promoter include the G-box.

The alcohol dehydrogenase (Adh) gene of Arabidopsis is expressed constitutively in immature seedlings and cells in suspension, and may be induced by hypoxic stress only in roots of mature plants. Deletions and G-box mutations of the Adh promoter were assayed in Arabidopsis protoplasts by PEG-mediated transient expression. Sequence domains necessary for full gene activity are confined to the 384 bp immediately 5' to the transcription start site, and deletion to -177 results in greater than 90% reduction in promoter activity. Site-specific mutations of G-box bases result in greater than 60% reduction in activity and disrupt G-box factor binding in vitro.

Alcohol Dehydrogenase↗

PubMed1995

A GCC element and a G-box motif participate in ethylene-induced expression of the PRB-1b gene.

The PRB-1b gene codes for a basic-type pathogenesis-related protein and is activated at the transcriptional level by the plant hormone ethylene. To identify cis-acting DNA elements essential for ethylene induction, deleted and mutant forms of the PRB-1b promoter, fused to the beta-glucuronidase (GUS) coding region, were introduced in transgenic tobacco plants. A 73 bp fragment (X1 region) of the PRB-1b promoter, located between positions -213 and -141, was sufficient to confer ethylene responsiveness to the reporter gene. The X1 region contains a TAAGAGCCGCC motif (GCC-box) well conserved in several ethylene-inducible genes. A substitution mutation in this sequence, in the context of a 213 bp PRB-1b promoter, completely abolished ethylene induction in transgenic tobacco, defining this conserved motif as part of a cis-acting element responsive to ethylene. Three other mutations in the X1 region caused a pronounced decrease in the PRB-1b promoter activity in transgenic plants, but did not affect ethylene inducibility. One of them, localized in a G-box like motif (CACGTG), disrupted the binding site for a nuclear factor, as observed in gel-shift analysis. Interestingly, the mobility of the complex formed on the G-box element was dependent on its phosphorylation state. These results suggest that a cis-acting element involved in the perception of the ethylene signal resides in a GCC motif and acts in concert with additional elements in the regulation of ethylene-induced PRB-1b expression.

Base Sequence↗

PubMed1994

Purification and characterization of CopR, a transcriptional activator protein that binds to a conserved domain (cop box) in copper-inducible promoters of Pseudomonas syringae.

The copper resistance (cop) operon promoter (Pcop) of Pseudomonas syringae is copper-inducible, and requires the regulatory genes copRS. Sequence analysis revealed that CopR has significant homology with other known activator proteins from bacterial two-component regulatory systems. In the present study we characterized Pcop and its interaction with CopR. We found that crude protein extracts from copper-resistant and -sensitive strains of P. syringae contain a Pcop-specific DNA-binding protein. We hypothesized that this DNA-binding protein was the product of copR. A 27-kDa protein, which corresponded to the predicted copR product, was expressed from this gene in Escherichia coli. CopR was purified, and the first eight amino acids were sequenced to confirm its relationship to copR. Specific binding of purified CopR to the plasmid-borne Pcop and the chromosomally encoded cop homolog promoter (PcopH), identified in this report, was demonstrated using specific and non-specific promoter competitors in DNA mobility shift assays. DNAse I footprinting identified a conserved CopR binding region (cop box) on Pcop and PcopH. The cop box contains an inverted repeat within a stretch of 16 bp, which shares approximately 75% identity with the PhoB binding region from several phosphate regulon gene promoters in E. coli. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of copA, and the transcriptional initiation site of PcopH 88 bp 5' to the translational start site of the chromosomal homolog of copA. The cop box was localized to between positions -54 and -35 relative to the transcriptional initiation site of Pcop and PcopH. Deletion analysis of Pcop delimited copper-inducible activity to a 104-bp region. Pcop and PcopH do not share a sequence consensus with other characterized promoters from P. syrinagae or E. coli. The results presented delineate important regions on two copper-inducible promoters form P. syringae.

Bacterial Outer Membrane Proteins↗

PubMed1994

The glycinin box: a soybean embryo factor binding motif within the quantitative regulatory region of the 11S seed storage globulin promoter.

The soybean embryo factor binding sequence in the glycinin A2B1a gene promoter was delimited to an A/T-rich 9 bp sequence, 5'-TAATAATTT-3', designated as the glycinin box, by DNA footprinting and gel mobility shift assay using synthetic oligonucleotides. It was shown that the interaction with the factor takes place at a defined DNA sequence rather than at random A/T-rich sequence blocks in the glycinin 5' flanking region. There are four glycinin boxes in the quantitative regulatory region between positions -545 and -378 of the glycinin A2B1a promoter. Multiple nonamer motifs similar to the glycinin box were also found in the equivalent regions of other glycinin and legumin promoters, suggesting that they must be conserved as a binding site for the embryo factor that activates the differential and stage-specific expression of seed 11S globulin genes in leguminous plants.

Base Sequence↗

PubMed1989

Mutational analysis of the Bradyrhizobium japonicum common nod genes and further nod box-linked genomic DNA regions.

By insertional and deletional marker replacement mutagenesis the common nod region of Bradyrhizobium japonicum was examined for the presence of additional, essential nodulation genes. An open reading frame located in the 800 bp large intergenic region between nodD1 and nodA did not appear to be essential for nodulation of soybean. Furthermore, a strain with a deletion of the nodI- and nodJ-like genes downstream of nodC had a Nod+ phenotype. A mutant with a 1.7 kb deletion immediately downstream of nodD1 considerably delayed the onset of nodulation. This region carried a second copy of nodD (nodD2). A nodD1-nodD2 double mutant had a similar phenotype to the nodD2 mutant. Using a 22-mer oligonucleotide probe partially identical to the nod box sequence, a total of six hybridizing regions were identified in B. japonicum genomic DNA and isolated from a cosmid library. Sequencing of the hybridizing regions revealed that at least three of them represented true nod box sequences whereas the others showed considerable deviations from the consensus sequence. One of the three nod box sequences was the one known to be associated with nodA, whereas the other two were located 60 to 70 kb away from nif cluster I. A deletion of one of these two sequences plus adjacent DNA material (mutant delta 308) led to a reduced nodulation on Vigna radiata but not on soybean. Thus, this region is probably involved in the determination of host specificity.

Base Sequence↗

PubMed1984

Transparent box enables three dimensional viewing of CT imaging of the liver.

We designed a transparent box to reconstruct cross sectional computed tomography (CT) imagings of the liver into a three dimensional form. The objective was to clearly visualize the area of lesion and to facilitate clinical teaching. This box (31.5 X 22.0 X 18.5 cm) is made of acrylic board containing 16 demountable panels layered horizontally at 10-mm intervals. The transparency of the CT film is projected onto these panels at each height of the liver, and findings including location of the tumor, main vessels and adjacent organs are outlined with an erasable marker. After completion of each tracing, the panels are replaced in the box and the total layer forms a completed picture of the CT findings. Since 1981, we have used this tool to assess resectability and/or to select adequate procedures for treating 54 patients with liver tumor. The solid picture gives an definite location of the tumor and its extension. In the clinical work-up, indication for surgery and a procedure of choice can be discussed by the team, using this visual aid.

Humans↗