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PubMed1977

The physiological costs of box lifting.

Thirteen male subjects lifted boxes of three different weights for 20 min periods each. Ventilation, oxygen uptake and heart rates were measured. Each subject made subjective estimates of the degree of difficulty of the work he was doing. Oxygen uptake and ventilation were found to reach steady state after about four minutes and remain there for the rest of the work session. Heart rate for the two lighter boxes reached steady state after about thirteen minutes while the heart rate continued to rise when lifting was done with the heaviest box. The relationships between oxygen uptake, heart rate and work load tended to be curvilinear, whereas the ventilation was linear when compared to work load. The heart rate and oxygen uptake relationship was also curvilinear, while ventilation and oxygen uptake had a linear relationship. The subjective ratings were expected to be about one-tenth of the heart rate. Only the ratings made while lifting the lightest box reflected this. The heavier boxes were rated as being more difficult to lift than 10 times the heart rate per minute. The mechanical efficiencies were found to range between 4.4% and 6.2%.

Adult↗

PubMed1980

New brain-cutting box: its use in computerized axial tomographic scan and autopsy brain section correlation.

A new brain-cutting box aids in the sectioning of autopsied brains along planes that correspond to computerized axial tomographic (CT) scans levels. There are two plastic boxes, one inside the other. The inner one has a platform slanted at a 15 degree angle from the horizontal plane. The brain, resting on this platform, can be raised by elevating the inner box and can be sectioned along the horizontal upper free edges of the outer box. The resulting sections, cut at appropriate intervals, correspond to CT scans. This box has numerous advantages over existing brain-cutting devices.

Autopsy↗

PubMed1993

The sequence-specific high mobility group 1 box of TCF-1 adopts a predominantly alpha-helical conformation in solution.

The High Mobility Group (HMG) 1 box is a protein motif that mediates DNA binding in a novel family of transcription-regulating proteins. Several members of this family, including the lymphoid-specific proteins TCF-1 and LEF-1 and the mammalian sex-determining factor SRY, carry a single HMG box with affinity for the minor groove of the heptamer motif AACAAAG or variations thereof. To initiate studies on the structural characteristics of the TCF-1 HMG box, we have expressed the 87-amino acid HMG box in milligram quantities in Escherichia coli and purified the soluble peptide to > 95% homogeneity. The peptide bound DNA with the same specificity as the complete protein and was capable of inducing DNA bending. Circular dichroism (CD) analysis revealed the TCF-1 HMG box to adopt an approximately 60% alpha-helix/40% random coil conformation in solution. In the presence of an equimolar amount of double-stranded DNA containing the cognate motif, the CD spectrum changed significantly, implying the induction of a structural modification upon DNA/protein association.

Amino Acid Sequence↗

PubMed1996

A controlled comparison of light box and head-mounted units in the treatment of seasonal depression.

BACKGROUND: Patterns of response to the light box and head-mounted unit (HMUs) in seasonal affective disorder (SAD) appear to differ. The current study employed a "no light" condition to compare the response rates with the light box and HMU against a plausible placebo. METHOD: Forty-three subjects with DSM-III-R nonpsychotic, unipolar major depression, seasonal subtype, were randomly assigned, in a double-blind manner, to receive 2 weeks of active treatment with a light box (N=9) or HMU (N=12) that emitted no visible light, or 2 weeks of placebo treatment with a light box (N=12) or HMU (N=10) that emitted no visible light. Response was defined as a 50% or greater reduction in both the 17-item "typical" score and 8-item "atypical" score on the Structured Interview Guide for the Hamilton Rating Scale for Depression-SAD version (SIGH-SAD). RESULTS: Using ANOVA for repeated measures, with change in total SIGH-SAD score as the dependent measure, we found no significant main effect of light (F=0.20, p=N.S.) or unit (F=0.50, p=N.S.), and no interaction (F=0.21, p=N.S.). Using log-linear analysis, we found no significant difference in response rate between the four cells (likelihood ratio chi-square = 2.1, p=N.S.). Using chi-square analysis, we found no significant difference in response rates between patients who received light (48%) versus patients who received no light (41%; chi-square = 0.2, p=N.S.) or between patients who received the light box (38%) versus HMU (50%; chi-square = 0.62, p=N.S.). CONCLUSION: The failure to detect any significant difference in efficacy between active and placebo treatments calls into question the specificity of light in light therapy for SAD. Methodological limitations, particularly small sample size, are discussed.

Adult↗

PubMed1999

Transcription of the bone sialoprotein gene is stimulated by v-Src acting through an inverted CCAAT box.

Bone sialoprotein (BSP) is an early marker of differentiated osteoblasts that has been implicated in the nucleation of hydroxyapatite crystal formation during de novo bone formation. Although essentially specific to mineralizing connective tissues, BSP is also expressed ectopically by carcinomas that exhibit microcalcification and which metastasize to bone with high frequency. However, it is not known how BSP is regulated in transformed cells. Because the v-src oncogene induces expression of a number of genes that are involved in tumor growth and metastasis, including osteopontin, we have studied the effects of v-Src on transcription of the BSP gene. Transfection of mouse src-/- cells with a v-src expression vector increased the transcriptional activity of rat BSP promoter/luciferase chimeric constructs approximately 5-fold. Deletion analysis revealed that the v-Src activity was targeted to an inverted CCAAT box located immediately upstream from an inverted TATA box in the BSP promoter. Although mutation of the CCAAT box diminished the basal transcription activity of the BSP promoter, the Src-induced stimulation was completely abolished. Gel mobility shift analysis identified four nuclear factors that bound to this region of the BSP promoter, two of which required an intact CCAAT sequence. Monoclonal antibodies identified nuclear factor-Y (NF-Y) as the principal nuclear factor that bound to the CCAAT box; the second factor (beta) showing strong binding only in short constructs containing the CCAAT sequence. Transcription analyses with a dominant negative NF-Y expression vector confirmed that NF-Y mediated the action of v-Src. These studies indicate that BSP gene expression in transformed cells can be up-regulated by Src kinase activity through a mechanism mediated by the NF-Y transcription factor, which targets an inverted CCAAT box in the BSP gene promoter.

3T3 Cells↗

PubMed1998

Solution structure of a TBP-TAF(II)230 complex: protein mimicry of the minor groove surface of the TATA box unwound by TBP.

General transcription factor TFIID consists of TATA box-binding protein (TBP) and TBP-associated factors (TAF(II)s), which together play a central role in both positive and negative regulation of transcription. The N-terminal region of the 230 kDa Drosophila TAF(II) (dTAF(II)230) binds directly to TBP and inhibits TBP binding to the TATA box. We report here the solution structure of the complex formed by dTAF(II)230 N-terminal region (residues 11-77) and TBP. dTAF(II)230(11-77) comprises three alpha helices and a beta hairpin, forming a core that occupies the concave DNA-binding surface of TBP. The TBP-binding surface of dTAF(II)230 markedly resembles the minor groove surface of the partially unwound TATA box in the TBP-TATA complex. This protein mimicry of the TATA element surface provides the structural basis of the mechanism by which dTAF(II)230 negatively controls the TATA box-binding activity within the TFIID complex.

Amino Acid Sequence↗

PubMed2002

A regulated two-step mechanism of TBP binding to DNA: a solvent-exposed surface of TBP inhibits TATA box recognition.

The TATA box binding protein TBP plays a universally important role in eukaryotic nuclear transcription. By mutagenesis, we have discovered a solvent-exposed surface of the structured TBP core domain that is important for inhibition of the DNA binding and DNA-bending activities of full-length wild-type TBP. Full-length wild-type TBP initially binds the TATA box to form an unstable complex containing unbent DNA, and then it slowly forms a stable complex containing bent DNA. TFIIB greatly accelerates formation of a bent TFIIB-TBP-TATA box complex, and the inhibitory DNA binding surface of TBP contributes to the cooperativity of binding to TFIIB. Using TBP and TFIIB, we show that TBP can bind the TATA box through a regulated two-step mechanism, involving a transition from unbent complex to bent complex.

DNA↗

PubMed1996

The TATA box binding protein.

The TATA box binding protein is required by all three eukaryotic RNA polymerases to correctly initiate the transcription of ribosomal, messenger, small nuclear and transfer RNAs. Since the first gene encoding a TATA box binding protein was cloned from Saccharomyces cerevisiae, it has been the object of considerable biochemical and genetic study. Substantial progress has recently been made on structural and mechanistic studies of the protein. Three-dimensional structures newly elucidated include two TATA box binding proteins alone and bound to distinct TATA elements, and the ternary complex of transcription factor IIB recognizing a TATA box binding protein bound to a TATA element.

Animals↗

PubMed2001

Use of a combined enzymatic digestion/ESI mass spectrometry assay to study the effect of TATA-binding protein on photoproduct formation in a TATA box.

Recently, it was reported that TATA-binding protein (TBP) enhances (6-4) photoproduct formation in a TATA box under UVC irradiation [Aboussekhra and Thoma (1999) EMBO J. 18, 433-443]. The conclusions of that study were based on an indirect enzymatic assay that was not specific for (6-4) photoproducts. Herein we report the use of a recently developed coupled enzymatic digestion/mass spectrometry assay [Wang et al. (1999) Chem. Res. Toxicol. 12, 1077-1082] to identify unambiguously and quantify the photoproducts formed in a TATA box-containing dodecamer duplex sequence in the presence or absence of TBP binding. Exposure of the adenovirus major late promoter TATA box to a high dose of UVC irradiation in the absence of the C-terminal domain of yeast TBP leads to predominant formation of the cis-syn dimer within the T(4) tract, whereas exposure in the presence of TBP leads to almost exclusive formation of the (6-4) photoproduct. In contrast, the (6-4) product is not detected at high doses of UVB irradiation in the absence of TBP but is detected in the presence of TBP, although the cis-syn product predominates. When the products of UVB irradiation were subsequently exposed to a high dose of UVC irradiation in the presence of TBP, the (6-4) photoproduct again becomes nearly the exclusive photoproduct, indicating that the cis-syn dimer is being reversed to TT by UVC light. Both cis-syn and (6-4) photoproducts are formed in approximately equal amounts upon irradiation with small doses of UVC in the presence of TBP, but the fraction of (6-4) photoproduct increases with dose. Through the use of a TATA box containing a site-specifically deuterated thymine, it was found that (6-4) photoproducts formed most selectively at the second and third positions of the T(4) tract upon either UVB or UVC irradiation in the presence of TBP. By using the same substrate, it was found that UVC-induced TA formation was inhibited by TBP binding and that TA formation was greatest at the 5' end of the TATA sequence.

Amino Acid Sequence↗

PubMed2004

cDNA cloning and expression analysis of a novel human F-box domain containing gene.

F-box proteins are a large family of eukaryotic proteins that are characterized by an approximately 40 amino acid motif. Some F-box proteins are critical for the controlled degradation of cellular regulatory proteins. Here we report that a novel member of F-box proteins, FBXO35 gene, was cloned and identified during the large-scale sequencing analysis from a human fetal brain cDNA library. FBXO35 gene shares amino acid similarity with several putative mouse genes not only in F-box domain but also in the rest of the sequence, which indicates that FBXO35 might also contain some other unknown conserved domain. RT-PCR analysis indicated that FBXO35 gene had a ubiquitously low expression pattern in most human adult tissues. According to bioinformatics analysis, the FBXO35 gene was found located in chromosome 3p21.

Amino Acid Sequence↗

PubMed2004

Phosphorylation-dependent degradation of c-Myc is mediated by the F-box protein Fbw7.

The F-box protein Skp2 mediates c-Myc ubiquitylation by binding to the MB2 domain. However, the turnover of c-Myc is largely dependent on phosphorylation of threonine-58 and serine-62 in MB1, residues that are often mutated in cancer. We now show that the F-box protein Fbw7 interacts with and thereby destabilizes c-Myc in a manner dependent on phosphorylation of MB1. Whereas wild-type Fbw7 promoted c-Myc turnover in cells, an Fbw7 mutant lacking the F-box domain delayed it. Furthermore, depletion of Fbw7 by RNA interference increased both the abundance and transactivation activity of c-Myc. Accumulation of c-Myc was also apparent in mouse Fbw7-/- embryonic stem cells. These observations suggest that two F-box proteins, Fbw7 and Skp2, differentially regulate c-Myc stability by targeting MB1 and MB2, respectively.

Animals↗

PubMed2006

Regulation of mitochondrial fusion by the F-box protein Mdm30 involves proteasome-independent turnover of Fzo1.

Mitochondrial morphology depends on balanced fusion and fission events. A central component of the mitochondrial fusion apparatus is the conserved GTPase Fzo1 in the outer membrane of mitochondria. Mdm30, an F-box protein required for mitochondrial fusion in vegetatively growing cells, affects the cellular Fzo1 concentration in an unknown manner. We demonstrate that mitochondrial fusion requires a tight control of Fzo1 levels, which is ensured by Fzo1 turnover. Mdm30 binds to Fzo1 and, dependent on its F-box, mediates proteolysis of Fzo1. Unexpectedly, degradation occurs along a novel proteolytic pathway not involving ubiquitylation, Skp1-Cdc53-F-box (SCF) E3 ubiquitin ligase complexes, or 26S proteasomes, indicating a novel function of an F-box protein. This contrasts to the ubiquitin- and proteasome-dependent turnover of Fzo1 in alpha-factor-arrested yeast cells. Our results therefore reveal not only a critical role of Fzo1 degradation for mitochondrial fusion in vegetatively growing cells but also the existence of two distinct proteolytic pathways for the turnover of mitochondrial outer membrane proteins.

F-Box Proteins↗

PubMed1993

Targeting TBP to a non-TATA box cis-regulatory element: a TBP-containing complex activates transcription from snRNA promoters through the PSE.

In the human small nuclear RNA (snRNA) promoters, the presence of a TATA box recognized by the TATA box-binding protein (TBP) determines the selection of RNA polymerase III over RNA polymerase II. The RNA polymerase II snRNA promoters are, therefore, good candidates for TBP-independent promoters. We show here, however, that TBP activates transcription from RNA polymerase II snRNA promoters through a non-TATA box element, the snRNA proximal sequence element (PSE), as part of a new snRNA-activating protein complex (SNAPc). In contrast to the previously identified TBP-containing complexes SL1, TFIID, and TFIIIB, which appear dedicated to transcription by a single RNA polymerase, SNAPc is also essential for RNA polymerase III transcription from the U6 snRNA promoter. The U6 initiation complex appears to contain two forms of TBP, one bound to the TATA box and one bound to the PSE as a part of SNAPc, suggesting that multiple TBP molecules can have different functions within a single promoter.

Animals↗

PubMed1995

The symmetry of the yeast U6 RNA gene's TATA box and the orientation of the TATA-binding protein in yeast TFIIIB.

The central RNA polymerase III (Pol III) transcription factor TFIIIB is composed of the TATA-binding protein (TBP), Brf, a protein related to TFIIB, and the product of the newly cloned TFC5 gene. TFIIIB assembles autonomously on the upstream promoter of the yeast U6 snRNA (SNR6) gene in vitro, through the interaction of its TBP subunit with a consensus TATA box located at base pair -30. As both the DNA-binding domain of TBP and the U6 TATA box are nearly twofold symmetrical, we have examined how the binding polarity of TFIIIB is determined. We find that TFIIIB can bind to the U6 promoter in both directions, that TBP is unable to discern the natural polarity of the TATA element and that, as a consequence, the U6 TATA box is functionally symmetrical. A modest preference for TFIIIB binding in the natural direction of the U6 promoter is instead dictated by flanking DNA. Because the assembly of TFIIIB on the yeast U6 gene in vivo occurs via a TFIIIC-dependent mechanism, we investigated the influence of TFIIIC on the binding polarity of TFIIIB. TFIIIC places TFIIIB on the promoter in one direction only; thus, it is TFIIIC that primarily specifies the direction of transcription. Experiments using TFIIIB reconstituted with the altered DNA specificity mutant TBPm3 demonstrate that in the TFIIIB-U6 promoter complex, the carboxy-terminal repeat of TBP contacts the upstream half of the TATA box. This orientation of yeast TBP in Pol III promoter-bound TFIIIB is the same as in Pol II promoter-bound TFIID and in TBP-DNA complexes that have been analyzed by X-ray crystallography.

Base Sequence↗

PubMed2006

Targeted silencing of Jab1/Csn5 in human cells downregulates SCF activity through reduction of F-box protein levels.

BACKGROUND: SCF ubiquitin ligases target numerous proteins for ubiquitin dependent proteolysis, including p27 and cyclin E. SCF and other cullin-RING ligases (CRLs) are regulated by the ubiquitin-like protein Nedd8 that covalently modifies the cullin subunit. The removal of Nedd8 is catalyzed by the Jab1/MPN domain metalloenzyme (JAMM) motif within the Csn5 subunit of the Cop9 Signalosome. RESULTS: Here, we conditionally knock down Csn5 expression in HEK293 human cells using a doxycycline-inducible shRNA system. Cullin levels were not altered in CSN-deficient human cells, but the levels of multiple F-box proteins were decreased. Molecular analysis indicates that this decrease was due to increased Cul1- and proteasome-dependent turnover. Diminished F-box levels resulted in reduced SCF activity, as evidenced by accumulation of two substrates of the F-box protein Fbw7, cyclin E and c-myc, in Csn5-depleted cells. CONCLUSION: We propose that deneddylation of Cul1 is required to sustain optimal activity of SCF ubiquitin ligases by repressing 'autoubiquitination' of F-box proteins within SCF complexes, thereby rescuing them from premature degradation.

COP9 Signalosome Complex↗

PubMed1996

Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development.

A novel family of genes, characterized by the presence of a region of homology to the DNA-binding domain of the Brachyury (T) locus product, has recently been identified. The region of homology has been named the T-box, and the new mouse genes that contain the T-box domain have been named T-box 1-6 (Tbx1 through Tbx6). As the basis for further study of the function and evolution of these genes, we have examined the expression of 5 of these genes, Tbx1-Tbx5, across a wide range of embryonic stages from blastocyst through gastrulation and early organogenesis by in situ hybridization of wholemounts and tissue sections. Tbx3 is expressed earliest, in the inner cell mass of the blastocyst. Four of the genes are expressed in different components of the mesoderm or mesoderm/endoderm during gastrulation (Tbx1 and Tbx3-5). All of these genes have highly specific patterns of expression during later embryogenesis, notably in areas undergoing inductive tissue interactions. In several cases there is complementary expression of different genes in 2 interacting tissues, as in the lung epithelium (Tbx1) and lung mesenchyme (Tbx2-5), and in mammary buds (Tbx3) and mammary stroma (Tbx2). Tbx1 shows very little overlap in the sites of expression with the other 4 genes, in contrast to a striking similarity in expression between members of the 2 cognate gene sets, Tbx2/Tbx3 and Tbx4/Tbx5. This is a clear reflection of the evolutionary relationship between the 5 genes since the divergence of Tbx1 occurred long before the relatively recent divergence of Tbx2 and 3 and Tbx4 and 5 from common ancestral genes. These studies are a good indication that the T-box family of genes has important roles in inductive interactions in many stages of mammalian embryogenesis.

Animals↗

PubMed1996

Localization of the 12-O-tetradecanoylphorbol-13-acetate response of the human ornithine decarboxylase promoter to the TATA box.

In a previous study, we narrowed the region of the human ornithine decarboxylase (ODC) promoter responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA) to nt -42 to +54 around the transcription initiation site (Kim YJ, Pan H, Verma AK, Mol Carcinog 10:169-179, 1994). Here we report defining the role of the TATA box in TPA-induced transcription from the -42/+54 ODC promoter fragment. A transversion mutation at the third position of the TATA box (TATAAGT-->TAAAAGT) reduced TPA responsiveness of the reporter construct -42/+54 ODC-Luc by 49%. Electrophoretic mobility shift assays (EMSAs) using HeLa cell nuclear protein extracts revealed no differences in the binding pattern between the natural -42/+54 ODC promoter element and the -42/+54 ODC promoter element containing the T-->A mutation. However, antibodies to the general transcription factor TFIIB disrupted the DNA-protein complexes normally formed with the -42/+54 ODC promoter element in EMSAs. A consensus TATA box oligonucleotide formed two bands, with the faster mobility band displaying enhanced binding with nuclear protein extracts from TPA treated cells. Furthermore, incubation of HeLa cell nuclear extracts with an oligonucleotide containing the ODC TATA box also caused formation of two specific bands in EMSA. Both bands exhibited augmented binding to nuclear proteins from TPA-treated cells. Introduction of the T-->A transversion mutation in the ODC TATA oligonucleotide eliminated binding of the faster migrating band formed with the natural ODC TATA oligonucleotide. These results indicate that TPA modulation of the general transcription machinery may play a role in the TPA-activated transcription of the human ODC promoter.

Cell Nucleus↗

PubMed2004

T-box genes in early embryogenesis.

The T-box gene family, encoding related DNA-binding transcriptional regulators, plays an essential role in controlling many aspects of embryogenesis in a wide variety of organisms. The T-box genes exhibit diverse patterns of spatial and temporal expression in the developing embryo, and both genetic and molecular embryological studies have demonstrated their importance in regulating cell fate decisions that establish the early body plan, and in later processes underlying organogenesis. Despite these studies, little is known of either the regulation of the T-box genes or the identities of their transcriptional targets. The aim of this review is to examine the diverse yet conserved roles of several T-box genes in regulating early patterning in chordates and to discuss possible mechanisms through which this functional diversity might arise. Developmental Dynamics 229:201-218, 2004.

Amino Acid Sequence↗