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Sequences flanking the hexameric G-box core CACGTG affect the specificity of protein binding.

The CACGTG G-box motif is a highly conserved DNA sequence that has been identified in the 5' upstream region of plant genes exhibiting regulation by a variety of environmental signals and physiological cues. Gel mobility shift assays using a panel of G-box oligonucleotides differing in their flanking sequences identified two types of binding activity (A and B) in a cauliflower nuclear extract. Competition gel retardation assays demonstrated that the two types of binding activity were distinct. Type A binding activity interacted with oligonucleotides designated as class I elements, whereas type B binding activity interacted strongly with class II elements and weakly with class I elements. A third class of elements, null elements, did not exhibit any detectable binding under our assay conditions. Gel retardation analysis of nonpalindromic hybrid G-box oligonucleotides indicated that hybrid elements of the same class exhibited binding affinity commensurate with the affinity of the weaker element, hybrid class I/II elements exhibited only type B binding, and hybrid class I/null and class II/null elements did not show any detectable binding activity. These binding activities can be explained by the affinity of bZip G-box binding homo- or heterodimer subunits for G-box half sites. These experiments led to a set of classification rules that can predict the binding activity of all reported plant G-box motifs containing the consensus hexameric core. Tissue- and/or development-specific expression of genes containing G-box motifs may be regulated by the affinity of G-box proteins for the different classes of G-box elements.

Base Sequence↗

Studies of the Bradyrhizobium japonicum nodD1 promoter: a repeated structure for the nod box.

Induction of nod genes in Rhizobium and Bradyrhizobium species is dependent on the presence of plant-produced flavonoids, the NodD protein, and the cis-acting nod box promoter sequence. Although the nodD (nodD1) gene in Rhizobium species is constitutively expressed, nodD1 expression in Bradyrhizobium japonicum is inducible by isoflavones in a manner similar to that of the nodYABC operon. A consensus nod box sequence is found 5' of the nodYABC operon, whereas a presumptive, nod box-like sequence is found 5' of the nodD1 gene. As an initial step toward examining the nodD1 promoter, the transcriptional start sites of the nodD1 and nodYABC operons were determined and found to be 44 and 28 bp, respectively, downstream of their respective nod box sequences. A series of deletions of the nodD1 promoter were constructed and fused to the lacZ gene. Analysis of the activity of these deletions clearly showed that the divergent nod box sequence was essential for nodD1 induction by isoflavones or soybean seed extract. The induction of nodD1 expression requires NodD1, as tested in B. japonicum and in a heterologous system, Agrobacterium tumefaciens. On the basis of these data, we analyzed the published nod box sequences and propose a new consensus sequence composed of paired 9-bp repeats. Analysis of the nodD1 nod box and synthetic constructs of the nocYABC nod box indicate that at least two 9-bp repeats are required for NodD1-mediated induction. Furthermore, insertions between the paired repeats of the nodYABC nod box suggest that orientation of the repeats on opposite faces of the DNA helix is essential for maximum nod gene expression.

Amino Acid Sequence↗

Importance of the position of TYR R boxes for repression and activation of the tyrP and aroF genes in Escherichia coli.

Tyrosine-mediated repression of aroF and tyrP was studied by inserting DNA sequences between the two adjacent TYR R boxes which, in each case, overlap the respective RNA polymerase binding sites of these genes. In both cases, repression was greatest when homologous regions of these two TYR R boxes were on the same face of the DNA helix and the boxes were directly adjacent. An insertion of 3 bases was sufficient to abolish repression, which was reestablished as the boxes became separated by one full turn of the helix. These observations, coupled with the results of in vitro DNase I protection studies, supported the hypothesis that the binding of TyrR protein to the downstream boxes required cooperative interaction with TyrR protein already bound to the upstream boxes. In the case of tyrP, moving the upstream box also affected activation. Maximal activation was observed when the box was moved 3 or 12 to 14 residues upstream. Practically no activation was seen at intermediate positions, such as +7 and -4. It is hypothesized that these results indicate positions allowing maximal interaction between TyrR protein bound to the upstream box and RNA polymerase bound to the RNA polymerase binding site.

Base Sequence↗

Importance of a flanking AT-rich region in target site recognition by the GC box-binding zinc finger protein MIG1.

MIG1 is a zinc finger protein that mediates glucose repression in the yeast Saccharomyces cerevisiae. MIG1 is related to the mammalian Krox/Egr, Wilms' tumor, and Sp1 finger proteins. It has two fingers and binds to a GCGGGG motif that resembles the GC boxes recognized by these mammalian proteins. We have performed a complete saturation mutagenesis of a natural MIG1 site in order to elucidate its binding specificity. We found that only three mutations within the GC box retain the ability to bind MIG1: G1 to C, C2 to T, and G5 to A. This result is consistent with current models for zinc finger-DNA binding, which assume that the sequence specificity is determined by base triplet recognition within the GC box. Surprisingly, we found that an AT-rich region 5' to the GC box also is important for MIG1 binding. This AT box is present in all natural MIG1 sites, and it is protected by MIG1 in DNase I footprints. However, the AT box differs from the GC box in that no single base within it is essential for binding. Instead, the AT-rich nature of this sequence seems to be crucial. The fact that AT-rich sequences are known to increase DNA flexibility prompted us to test whether MIG1 bends DNA. We found that binding of MIG1 is associated with bending within the AT box. We conclude that DNA binding by a simple zinc finger protein such as MIG1 can involve both recognition of the GC box and flanking sequence preferences that may reflect local DNA bendability.

Amino Acid Sequence↗

The clinical dementia rating sum of box score in mild dementia.

BACKGROUND: Making an early diagnosis of dementia is becoming increasingly important, but is difficult in practice. The Clinical Dementia Rating (CDR) scale is a widely used dementia staging instrument, yielding a global score and a summated score (sum of box score). This study examines the utility of the CDR sum of box score, rather than the CDR global score, in making a diagnosis of early dementia. OBJECTIVE: To determine whether the CDR sum of box score is predictive of an ICD-10 diagnosis of dementia in cases with mild cognitive deficits. METHODS: Clinical data recorded on our Memory Clinic database were examined for all patients seen over a 6-year period. Data were extracted from 276 first visits in which patients had scored 0.5 using the CDR global score. We examined the relationship between CDR sum of box score and consensus diagnosis of dementia using logistic regression. RESULTS: We found that increased CDR sum of box score was significantly associated with a higher probability of being assigned an ICD-10 diagnosis of dementia (p < 0.001). The odds ratio for the coefficient of CDR sum of box was 2.3 (95% CI 1.7-3.1), indicating that the likelihood of being diagnosed as having dementia increased by a factor of 2.3 for every point increase on the CDR sum of box score. CONCLUSION: These findings indicate that the CDR sum of box score provides additional information to the CDR global score in mild cases. The CDR sum of box score is a helpful indicator in making/excluding a diagnosis of dementia in people with mild cognitive deficits.

Alzheimer Disease↗

DEK binding to class II MHC Y-box sequences is gene- and allele-specific.

Using electrophoretic mobility shift assays, we examined sequence-specific binding of DEK, a potential autoantigen in juvenile rheumatoid arthritis, to conserved Y-box regulatory sequences in class II MHC gene promoters. Nuclear extracts from several cell lines of different phenotypes contained sequence-specific binding activity recognizing DRA, DQA1*0101, and DQA1*0501 Y-box sequences. Participation of both DEK and NF-Y in the DQA1 Y-box binding complex was confirmed by 'supershifting' with anti-DEK and anti-NF-Y antibodies. Recombinant DEK also bound specifically to the DQA1*0101 Y box and to the polymorphic DQA1*0501 Y box, but not to the consensus DRA Y box. Measurement of the apparent dissociation constants demonstrated a two- to fivefold difference in DEK binding to the DQA1 Y-box sequence in comparison with other class II MHC Y-box sequences. Residues that are crucial for DEK binding to the DQA1*0101 Y box were identified by DNase I footprinting. The specific characteristics of DEK binding to these related sequences suggests a potential role for DEK in differential regulation of class II MHC expression, and thus in the pathogenesis of juvenile rheumatoid arthritis and other autoimmune diseases.

Alleles↗

The Bearded box, a novel 3' UTR sequence motif, mediates negative post-transcriptional regulation of Bearded and Enhancer of split Complex gene expression.

During the development of the Drosophila adult peripheral nervous system (PNS), inhibitory cell-cell interactions mediated by the Notch receptor are essential for proper specification of sensory organ cell fates. We have reported previously (M. W. Leviten, E. C. Lai and J. W. Posakony (1997) Development 124, 4039-4051) that the 3' untranslated regions (UTRs) of many genes involved in Notch signalling, including Bearded (Brd) and the genes of the Enhancer of split Complex (E(spl)-C), contain (often in multiple copies) two novel heptanucleotide sequence motifs, the Brd box (AGCTTTA) and the GY box (GTCTTCC). Moreover, the molecular lesion associated with a strong gain-of-function mutant of Brd suggested that the loss of these sequence elements from its 3' UTR might be responsible for the hyperactivity of the mutant gene. We show here that the wild-type Brd 3' UTR confers negative regulatory activity on heterologous transcripts in vivo and that this activity requires its three Brd box elements and, to a lesser extent, its GY box. We find that Brd box-mediated regulation decreases both transcript and protein levels, and our results suggest that deadenylation or inhibition of polyadenylation is a component of this regulation. Though Brd and the E(spl)-C genes are expressed in spatially restricted patterns in both embryos and imaginal discs, we find that the regulatory activity that functions through the Brd box is both temporally and spatially general. A Brd genomic DNA transgene with specific mutations in its Brd and GY boxes exhibits hypermorphic activity that results in characteristic defects in PNS development, demonstrating that Brd is normally regulated by these motifs. Finally, we show that Brd boxes and GY boxes in the E(spl)m4 gene are specifically conserved between two distantly related Drosophila species, strongly suggesting that E(spl)-C genes are regulated by these elements as well.

Animals↗

Improved staining boxes for fast, uniform staining of ultrathin sections on grids.

A standard LKB (LKB-Produkter Ab. S-161, 25 Brommma 1, Sweden) grid storage box is converted into several grid staining boxes by sawing the body of the box into segments along rows of its grid storage cavities. The staining boxes can be cut out to any required size or shape. The polymethyacrylate storage box cover is discarded. Covers for the staining boxes are cut from thin sheet vinyl, which is more chemically resistant than polymethyacrylate. Corresponding 2 mm diameter holes are drilled through the vinyl covers and the bottoms of the grid storage cavities of the staining boxes to convert the storage cavities into staining chambers. For staining, the covers are tied to the boxes with sewing thread and the assembled units are put into vials. The separate staining chambers prevent intermingling of and mechanical damage to grids during the staining procedure. Ultrathin sections are more cleanly and uniformly stained in bulk by the use of these staining boxes than they are when stained individually by a standard method.

Cytological Techniques↗

[Signal detectability of mammography depends on film-screen system and luminance of view box].

High-density film and the high-luminance view-box system are being recommended for mammograms owing to the improved detection of masses. However, this system causes an increase in radiation. Therefore, the purpose of this study was to assess whether the detection of masses would improve using the normal-luminance view box and normal-density film with different types of contrast systems. Low-contrast detection using ROC analysis and high-contrast detection using an ACR phantom were evaluated for the following systems: high-density film and high-luminance view box, normal-density film and normal-luminance view box, and normal-density film with wide latitude and normal-luminance view box. The results showed no significant variation in the detectability of the system with high-density film and high-luminance view box and the normal-density film with wide latitude and normal-luminance view box. However, in terms of low-contrast visibility, the system using normal-density film and normal-luminance view box was significantly reduced in comparison with the others. Therefore, the system with normal-density film with wide latitude and the normal-luminance view box is recommended because of reduced radiation dose.

Breast Neoplasms↗

Differences in DNA sequence specificity among MHC class II X box binding proteins.

The class II (Ia) MHC Ag are integral membrane proteins whose expression is limited to specific cell types. A pair of consensus sequences, X and Y, is found upstream from all class II genes and deletion of each of these sequences eliminates expression of transfected genes. Cells that express Ia demonstrate a coordinate response to lymphokines and other stimuli. These conserved sequences might, therefore, play a role in tissue specificity or lymphokine inducibility of Ia gene expression. The X box sequence of the murine class II A alpha gene diverges much more substantially from the X consensus than does the Y box motif of this gene. We demonstrate that this X box motif is nonetheless recognized by sequence-specific DNA-binding proteins, as is the more closely conserved Y box. Gel retardation assays and DNase I footprints were compared for a panel of Ia+ and Ia- cells as well as for cells stimulated with the Ia-inducing lymphokines IL-4 and IFN-gamma. The level, retardation pattern and region of DNA contact were comparable in all instances. Thus the availability of active DNA-binding X and Y box factors cannot alone account for the regulation of A alpha expression. To test whether the same set of proteins binds all class II MHC conserved motifs, oligonucleotide probe binding and cross-competition experiments with X box sequences from A alpha, E alpha, and E beta genes were performed. These studies demonstrated A alpha, E alpha, and E beta DNA-protein complexes with unique mobilities and specificities. In addition, all three X box oligonucleotide probes generated one faint complex with an affinity profile of E beta greater than E alpha much greater than A alpha. These three complexes comigrated and thus may represent a communal binding protein. The data are most consistent with the conclusion that multiple proteins bind class II MHC X boxes. For A alpha, the predominant complexes represent different specificities from the predominant E alpha and E beta X box binding proteins.

Animals↗

An inverted TATA box directs downstream transcription of the bone sialoprotein gene.

The orientation of the TATA box is thought to direct downstream transcription of eukaryotic genes by RNA polymerase II. However, the putative TATA box in the promoter of the bone sialoprotein (BSP) gene, which codes for a tissue-specific and developmentally regulated bone matrix protein, is inverted (5'-TTTATA-3') relative to the consensus TATA box sequence (5'-TATAAA-3') and is overlapped by a vitamin D3-response element. Here we show that the inverted TATA sequence in the rat BSP gene binds to recombinant TATA-box-binding protein (TBP) with an affinity similar to that observed with the consensus TATA box, and site-directed point mutations in the inverted TATA sequence (mutating TTTATA into TCTCTA) abrogate both TBP binding and BSP promoter activity. However, when the inverted TATA sequence is changed to a canonical TATAAA, the TBP- and vitamin D3 receptor-binding properties together with the BSP promoter activity are retained. In addition, we found that the TBP is required to reconstitute in vitro transcription driven by the BSP promoter. These studies, which have revealed a naturally occurring inverted TATA box that can bind TBP and direct downstream transcription, demonstrate that the orientation of the TATA box does not determine the direction of transcription in higher eukaryotic genes. Consequently, the inverted TATA box that is conserved in the human, rat and mouse BSP gene promoters will provide an excellent in vivo model to investigate the polarity of the transcription factor IID-DNA complex and its relation to downstream transcription.

Animals↗

RNA polymerase II/III transcription specificity determined by TATA box orientation.

The TATA box sequence in eukaryotes is located about 25 bp upstream of many genes transcribed by RNA polymerase II (Pol II) and some genes transcribed by RNA polymerase III (Pol III). The TATA box is recognized in a sequence-specific manner by the TATA box-binding protein (TBP), an essential factor involved in the initiation of transcription by all three eukaryotic RNA polymerases. We have investigated the recognition of the TATA box by the Pol II and Pol III basal transcription machinery and its role in establishing the RNA polymerase specificity of the promoter. Artificial templates were constructed that contained a canonical TATA box as the sole promoter element but differed in the orientation of the 8-bp TATA box sequence. As expected, Pol II initiated transcription in unfractionated nuclear extracts downstream of the "forward" TATA box. In distinct contrast, transcription that initiated downstream of the "reverse" TATA box was carried out specifically by Pol III. Importantly, this effect was observed regardless of the source of the DNA either upstream or downstream of the TATA sequence. These findings suggest that TBP may bind in opposite orientations on Pol II and Pol III promoters and that opposite, yet homologous, surfaces of TBP may be utilized by the Pol II and Pol III basal machinery for the initiation of transcription.

Animals↗

The amino-terminal portion of the F-box protein Met30p mediates its nuclear import and assimilation into an SCF complex.

SCF complexes are a conserved family of ubiquitin ligases composed of a common core of components and a variable component called an F-box protein that defines substrate specificity. The F-box motif links the F-box protein to the core components via its interaction with Skp1p. In yeast, the SCFMet30p complex contains the Met30p F-box protein and regulates Met4p, a transcription factor that mediates sulfur fixation and methionine biosynthesis. Although a nuclear protein, Met30p lacks a definable nuclear localization sequence. Here we show that the entire amino-terminal half of Met30p is required for its proper nuclear localization. Mutations in the F-box, but not mutations in Skp1p, affect Met30p nuclear localization, indicating that the F-box motif plays an important role in Met30p trafficking independent of its interaction with Skp1p binding. Met30p mutants that poorly localize to the nucleus display increased nuclear to cytoplasmic exchange, indicating that the amino terminus mediates nuclear retention in addition to nuclear import. The Met30p F-box motif, residues 180-225, is necessary and sufficient to bind Skp1p; however, mutations upstream of the Met30p F-box inhibit Skp1p binding. We propose that additional factors bind the amino-terminal region of Met30p and mediate its nuclear localization and assimilation into an SCF complex.

Active Transport, Cell Nucleus↗

Skp1p and the F-box protein Rcy1p form a non-SCF complex involved in recycling of the SNARE Snc1p in yeast.

Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin-ligases composed of a core complex including Skp1p, Cdc53p, Hrt1p, the E2 enzyme Cdc34p, and one of multiple F-box proteins which are thought to provide substrate specificity to the complex. Here we show that the F-box protein Rcy1p is required for recycling of the v-SNARE Snc1p in Saccharomyces cerevisiae. Rcy1p localized to areas of polarized growth, and this polarized localization required its CAAX box and an intact actin cytoskeleton. Rcy1p interacted with Skp1p in vivo in an F-box-dependent manner, and both deletion of its F box and loss of Skp1p function impaired recycling. In contrast, cells deficient in Cdc53p, Hrt1p, or Cdc34p did not exhibit recycling defects. Unlike the case for F-box proteins that are known to participate in SCF complexes, degradation of Rcy1p required neither its F box nor functional 26S proteasomes or other SCF core subunits. Importantly, Skp1p was the only major partner that copurified with Rcy1p. Our results thus suggest that a complex composed of Rcy1p and Skp1p but not other SCF components may play a direct role in recycling of internalized proteins.

Binding Sites↗

Neurologic aspects of boxing.

The assessment and prevention of potentially adverse neurologic consequences of boxing requires two important considerations. Acute neurologic injuries should be distinguished from chronic brain injuries and the level of competitive boxing (ie, amateur vs professional) must also be taken into account. Acute neurologic injuries such as concussion, post-concussion syndrome, intracranial hemorrhage, and brain contusion are more readily identified than chronic neurologic injuries because of their immediate devastation of the nervous system. In contrast, chronic neurologic injuries differ in their pathophysiologic mechanisms that are exemplified by an insidious onset and progression after the cessation of boxing. Accordingly, the chronic traumatic encephalopathy of boxing poses the most serious neurologic threat of boxing. Amateur boxing differs from professional boxing in the duration of fights, rules and regulatory policies, medical evaluation, and protective devices. These factors could produce a differential effect on the risk of injury to the brain. The prevention of neurologic injuries in boxing requires the integration of proper neurologic evaluation by qualified ring-side physicians, the design and utilization of effective protective devices, and the establishment of national regulatory agencies.

Athletic Injuries↗

F-box proteins: the key to protein degradation.

The eukaryotic protein degradation pathway involves the ubiquitin (Ub) modification of substrates targeted for degradation by the 26S proteasome. The addition of Ub, a process called ubiquitination, is mediated by enzymes including the E3 Ub ligases which transfer the Ub to targeted substrates. A major type of E3 Ub ligases, the SCF (Skp-Cullin-F-box) complex, is composed of four major components: Skp1, Cul1/Cdc53, Roc1/Rbx1/Hrt1, and an F-box protein. The F-box component of the SCF machineries is responsible for recognizing different substrates for ubiquitination. Interaction with components of the SCF complex is mediated through the F-box motif of the F-box protein while it associates with phosphorylated substrates through its second protein-protein interaction motif such as Trp-Asp (WD) repeats or leucine-rich repeats (LRRs). By targeting diverse substrates, F-box proteins exert controls over stability of proteins and regulate the mechanisms for a wide-range of cellular processes. Here we discuss the importance of F-box proteins by providing a general overview and examples of how F-box proteins function in various cellular settings such as tissue development, cell proliferation, and cell death, in the modeling organism Drosophila.

Animals↗

Transcriptional regulation of environmentally inducible genes in plants by an evolutionary conserved family of G-box binding factors.

1. In reviewing a number of the most intensely studied environmentally inducible promoters it becomes clear that the presence of two cis-acting elements are critical for promoter activity, one of which is the G-box (CCACGTGG). A mutation in one of the two elements abolishes or severely reduces the ability of the promoter to respond to environmental changes. The sequence of the second cis-acting element, positioned nearby the G-box, is not conserved among the different inducible promoters, but may be similar among promoters induced by the same signal. The spacing between the G-box and the second cis-acting element appears to be critical, suggesting a direct interaction between the respective binding factors. We speculate on a potential role of the G-box promoter element in the signal induction of promoter activity. 2. From a number of plant species nuclear proteins interacting with the G-box have been identified. Recently, G-box Binding Factors (GBF) have been isolated by screening cDNA expression libraries with a characterized G-box cis-acting element as DNA probe. The deduced amino acid sequence of the GBF clones revealed that they possess the features of the basic leucine zipper class of trans-acting factors. By amino acid sequence comparison and limited mutational analysis, we define amino acids critical for G-box binding specificity. All GBFs isolated to date have a conserved proline-rich domain involved in transcriptional activation. A number of GBFs are inducible by a particular environmental signal. 3. Recently, a protein designated GF14 has been isolated that is associated with the GBF protein complex. The protein has homology to mammalian brain specific proteins, which seem to function as regulators of phosphorylation events. GBF activity is regulated by phosphorylation. The GF14 proteins may therefore impose an additional control on gene expression.

Amino Acid Sequence↗

Boxing, youth and children.

Boxing remains a popular spectator sport, success in which is achieved by the promotion of boxing in the childhood years. Despite the failure to ban boxing generally, there remains the realistic expectation that organised boxing by children below the age of consent (appropriately 16 years) is still achievable. There are two reasons for banning children under the age of 16 years from boxing. The first is that children have little awareness of risk, specifically the risk of chronic encephalopathy, which develops only after a lag period measured in decades or more. The second is that there is no place in contemporary society for a youth sport which has, as its primary goal, the infliction of acute brain damage on an opponent. This paper analyses the medical and ethico-social issues implicit in this subject. Boxing, in historical perspective, has been an altruistic amateur sport for boys and male youths, and has a proud tradition. But in the context of social evolution, and in current perspective to prevent exploitation of underprivileged youths, the time has come for an absolute ban on underage boxing. Such is achievable even if adult boxing remains an Olympic and television sport for some time to come.

Adolescent↗