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PubMed1988

The YYRR box: a conserved dipyrimidine-dipurine sequence element in Drosophila and other eukaryotes.

We have discovered a novel DNA sequence element in Drosophila which is based upon a CTGA tandem repeat. This element has been named the YYRR box to emphasize its dipyrimidine-dipurine nature which is predicted to have unusual structural features. Southern hybridization analysis of genomic DNA indicates the presence of 25-30 copies of the YYRR box in each of three Drosophila species (melanogaster, pseudoobscura, and virilis) and conservation of genomic location within species. Similar analysis of human and rat DNA indicates the presence of YYRR related sequences in mammals as well. YYRR boxes have been localized to two genetic loci in Drosophila: Gld and a gene tentative identified as ted. These two genes exhibit correlated patterns of developmental expression and an identical mutant phenotype. Sequence analysis of the Gld YYRR box in three Drosophila species revealed a high degree of conservation despite its intronic location.

Animals↗

PubMed1994

The RNA polymerase I transcription factor UBF is a sequence-tolerant HMG-box protein that can recognize structured nucleic acids.

Upstream Binding Factor (UBF) is important for activation of ribosomal RNA transcription and belongs to a family of proteins containing nucleic acid binding domains, termed HMG-boxes, with similarity to High Mobility Group (HMG) chromosomal proteins. Proteins in this family can be sequence-specific or highly sequence-tolerant binding proteins. We show that Xenopus UBF can be classified among the sequence-tolerant class. Methylation interference assays using enhancer DNA probes failed to reveal any critical nucleotides required for UBF binding. Selection by UBF of optimal binding sites among a population of enhancer oligonucleotides with randomized sequences also failed to reveal any consensus sequence. The minor groove specific drugs chromomycin A3, distamycin A and actinomycin D competed against UBF for enhancer binding, suggesting that UBF, like other HMG-box proteins, probably interacts with the minor groove. UBF also shares with other HMG box proteins the ability to bind synthetic cruciform DNA. However, UBF appears different from other HMG-box proteins in that it can bind both RNA (tRNA) and DNA. The sequence-tolerant nature of UBF-nucleic acid interactions may accommodate the rapid evolution of ribosomal RNA gene sequences.

Animals↗

PubMed1994

Binding of Y-box proteins to RNA: involvement of different protein domains.

Eukaryotic Y-box proteins are reported to interact with a wide variety of nucleic acid structures to act as transcription factors and mRNA masking proteins. The modular structure of Y-box proteins includes a highly conserved N-terminal cold-shock domain (CSD, equivalent to the bacterial cold-shock proteins) plus four basic C-terminal domains containing arginine clusters and aromatic residues. In addition, the basic domains are separated by acidic regions which contain several potential sites for serine/threonine phosphorylation. The interaction of Y-box proteins, isolated from Xenopus oocytes (FRGY2 type), with RNA molecules has been studied by UV crosslinking and protein fragmentation. We have identified two distinct binding activities. The CSD interacts preferentially with the polypurines poly(A,G) and poly(G) but not poly(A), this activity being sensitive to 5 mM MgCl2 but not to 5 mM spermidine. In the presence of 1 mM MgCl2 or 1 mM spermidine, the basic domains interact preferentially with poly(C,U), this activity being sensitive to 0.5 M NaCl. Binding of the basic domains is also sensitive to low concentrations of heparin. The basic domains can be crosslinked individually to labelled RNA. These results are discussed with reference to the various specificities noted in the binding of Y-box proteins to RNA and DNA.

Animals↗

PubMed1994

Novel conserved sequence motifs in plant G-box binding proteins and implications for interactive domains.

The G-box is a cis-acting DNA sequence present in several plant promoters that are regulated by diverse signals such as UV irradiation, anaerobiosis, abscissic acid and light. Several basic/leucine zipper (bZIP) proteins from different plant species have been identified as high affinity G-box binding proteins. Although their capability to enhance transcription has been demonstrated, their precise function in transcriptional activation is still unknown. We have isolated three cDNAs from young tomato fruit that encode bZIP G-box binding proteins (GBF4, GBF9 and GBF12). They bind to the G-box sequence in the tomato rbcS1, rbcS2 and rbcS3A promoters. GBF9 binding resulted in a DNase I footprint identical to that obtained with tomato nuclear extract and different from the DNase I protection obtained with GBF4 and GBF12. The mRNAs of all three GBFs were most abundant in tomato fruit and seeds, moderately abundant in root and least abundant in leaves. Protein sequences outside of the bZIP domains were compared with the known GBFs from other plants and seven conserved motifs of seven to 35 amino acids length have been identified. Based on the presence of these motifs, three classes of GBFs can be defined that are conserved among plant species. GBF9, the predominantly expressed tomato GBF, is the first member of its class isolated from dicot plants. Three conserved motifs from two of the classes are highly hydrophilic and are predicted to be exposed on the surface of the proteins. These motifs likely define novel interactive domains in the different classes of GBFs that could provide a new tool to determine how distinct regulatory signals are transmitted through GBFs to activate transcription.

Base Sequence↗

PubMed1995

ZEMa, a member of a novel group of MADS box genes, is alternatively spliced in maize endosperm.

The identification of a number of cis-elements which direct gene expression in maize endosperm, and the characterization of corresponding DNA binding proteins, point to the interaction of different classes of transcription factors in this tissue. To assess whether MADS box genes are also involved in maize endosperm development, cDNA and genomic MADS box clones have been isolated. The three cDNA clones ZEM1, ZEM2 and ZEM3 were cloned from a maize endosperm cDNA library using a probe based on sequences conserved in plant MADS box genes. Further transcripts were cloned by RT-PCR experiments and designated ZEM4 and ZEM5. Analysis of the corresponding genomic clones led to the identification of the ZEM2 MADS box gene family, three members of which were characterized sharing 97% sequence identity in corresponding domains. 100% sequence identities between cDNA and one of the genomic clones, conserved exon-intron boundaries and the demonstration of in vivo splicing in a maize endosperm transient expression system, show that the transcripts ZEM1-5 are derived by alternative splicing of ZEMa, one ZEM2 member. The ZEMa transcripts are present in almost all maize tissues, but specific differentially spliced forms accumulate preferentially in maturing endosperm and leaf. The function of the ZEMa gene is discussed in the light of similarities in the expression pattern with members of the human MEF2/RSRF gene family.

Alternative Splicing↗

PubMed1998

EcoKI with an amino acid substitution in any one of seven DEAD-box motifs has impaired ATPase and endonuclease activities.

For type I restriction systems, recently determined nucleotide sequences predict conserved amino acids in the subunit that is essential for restriction but not modification (HsdR). The conserved sequences emphasize motifs characteristic of the DEAD-box family of proteins which comprises putative helicases, and they identify a new candidate for motif IV. We provide evidence based on an analysis of Eco KI which supports both the relevance of DEAD-box motifs to the mechanism of restriction and the new definition of motif IV. Amino acid substitutions within the newly identified motif IV and those in six other previously identified DEAD-box motifs, but not in the original motif IV, confer restriction-deficient phenotypes. We have examined the relevance of the DEAD-box motifs to the restriction pathway by determining the steps permitted in vitro by the defective enzymes resulting from amino acid substitutions in each of the seven motifs. Eco KI purified from the seven restriction-deficient mutants binds to an unmethylated target sequence and, in the presence of AdoMet, responds to ATP by undergoing the conformational change essential for the pathway of events leading to DNA cleavage. The seven enzymes have little or no ATPase activity and no endonuclease activity, but they retain the ability to nick unmodified DNA, though at reduced rates. Nicking of a DNA strand could therefore be an essential early step in the restriction pathway, facilitating the ATP-dependent translocation of DNA, particularly if this involves DNA helicase activity.

Adenosine Triphosphatases↗

PubMed2000

A novel snail-related transcription factor Smuc regulates basic helix-loop-helix transcription factor activities via specific E-box motifs.

Snail family proteins are zinc finger transcriptional regulators first identified in Drosophila which play critical roles in cell fate determination. We identified a novel Snail -related gene from murine skeletalmusclecells designated Smuc. Northern blot analysis showed that Smuc was highly expressed in skeletal muscle and thymus. Smuc contains five putative DNA-binding zinc finger domains in its C-terminal half. In electrophoretic mobility shift assays, recombinant zinc finger domains of Smuc specifically bound to CAGGTG and CACCTG E-box motifs (CANNTG). Because basic helix-loop-helix transcription factors (bHLH) bind to the same E-box sequences, we examined whether Smuc competes with the myogenic bHLH factor MyoD for DNA binding. Smuc inhibited the binding of a MyoD-E12 complex to the CACCTG E-box sequence in a dose-dependent manner and suppressed the transcriptional activity of MyoD-E12. When heterologously targeted to the thymidine kinase promoter as fusion proteins with the GAL4 DNA-binding domain, the non-zinc finger domain of Smuc acted as a transcriptional repressor. Furthermore, overexpression of Smuc in myoblasts repressed transactivation of muscle differentiation marker Troponin T. Thus, Smuc might regulate bHLH transcription factors by zinc finger domains competing for E-box binding, and non-zinc finger repressor domains might also confer transcriptional repression to control differentiation processes.

Amino Acid Sequence↗

PubMed2001

A MADS box gene from lily (Lilium Longiflorum) is sufficient to generate dominant negative mutation by interacting with PISTILLATA (PI) in Arabidopsis thaliana.

Lily MADS box gene 1 (LMADS1), with sequence homology to the AP3 family of genes, was cloned and characterized from lily (Lilium longiflorum). LMADS1 protein contains almost complete consensus sequence of the PISTILLATA (PI)-derived motif (YEFRVQPSQPNLH) found in the AP3 family of genes and paleoAP3 motif (YGSHDLRLA) found in the AP3 family of genes from the low eudicot, magnolid dicot and monocot species. LMADS1 mRNA was expressed in all four whorls of the flower and absent in the vegetative leaves. The LMADS1 protein was only detected in the petals and stamens, indicating that LMADS1 is possibly post-transcriptionally regulated in lily. Arabidopsis plants transformed with 35S::LMADS1 produced flowers with short petals and stamens, however, no floral organ conversion was observed. Ectopic expression of LMADS1 cDNA truncated with the MADS box domain in Arabidopsis generated the ap3-like dominant negative mutation in which the petals were converted into sepal-like structures and the stamens were converted into carpel-like structures. Yeast two-hybrid analysis indicated that LMADS1 truncated with the MADS box domain is able to sufficiently interact with the Arabidopsis PI protein. This result supports that LMADS1 is the functional counterpart of the AP3 gene in lily. Interestingly, in contrast to other B functional genes, LMADS1 truncated with the MADS box domain is able to strongly form homodimers. LMADS1 may represent an ancestral form of the B function gene, which retains the ability to form homodimers in regulating petal and stamen development in lily.

Amino Acid Sequence↗

PubMed1983

On the effect of a negative ion-generator and a mixing fan on the plate-out of radon decay products in a radon box.

Studies have been conducted on the effect of a negative ion-generator and a mixing fan on the radon daughter Working Level produced in an environmental radon "box". A dramatic decrease in the Working Level has been observed when either the ion-generator or the mixing fan is operating. The combined effect of the ion-generator and the mixing fan was substantially larger than that from either one of them alone. Experiments have been carried out over a wide range of relative humidity (45-95%) and at a temperature of 20 +/- 3 degrees C. The effects observed were nearly independent of relative humidity in the box. Preliminary experiments seem to indicate that the decrease in the Working Level is somewhat related to the possible build-up of electrostatic charge on the inner walls of the radon box. However, there is some difficulty in accepting charge build-up at high levels of relative humidity. To better understand the phenomena observed, a number of measurements are planned on the inner walls of the box. These include electrostatic charge, electric field, and measurement of alpha- and beta-activity.

Environmental Exposure↗

PubMed1992

Radiography of the carpal scaphoid. Experimental evaluation of "the carpal box" and first clinical results.

RATIONALE AND OBJECTIVES: The authors describe a new device ("the carpal box") for the radiographic detection of occult scaphoid fracture. METHODS: Fractures in the scaphoid of five cadaver specimens were mechanically produced. Subsequent examinations included conventional scaphoid radiography, multi-angle radiography, and radiography using the new device. The anatomic analysis of the specimens served as the standard for comparison. A pilot study was performed in six consecutive patients with suspected scaphoid fracture. RESULTS: Neither scaphoid radiography nor multi-angle radiography could confirm a fracture in two specimens, whereas all fractures were recognized on the carpal box radiographs. All scaphoid fractures were visualized by carpal box radiography, whereas scaphoid radiography was equivocal in one patient and negative in the other. CONCLUSIONS: Carpal box radiography may have additional value in the diagnosis of occult scaphoid fracture. This may lead to a reduction in costs and inconvenience for patients with clinically suspected scaphoid fracture and negative scaphoid radiography.

Carpal Bones↗

PubMed1992

Site-directed mutagenesis of the hydrogenase signal peptide consensus box prevents export of a beta-lactamase fusion protein.

A secretion vector, pVN1, expressing the [NiFe] hydrogenase signal peptide of Desulfovibrio vulgaris Hildenborough fused to beta-lactamase from Escherichia coli was constructed in order to study the unusual characteristics of hydrogenase signal peptides, which share a strictly conserved sequence, the consensus box: R-R-X-F-X-K. Although the hydrogenase signal peptide-beta-lactamase fusion protein was processed much more slowly than the fusion of beta-lactamase with its own signal peptide, the system mimicked several features expected for hydrogenase biosynthesis in E. coli, including increased export under anaerobic conditions. Site-directed mutagenesis of R(-28), the first arginine residue of the consensus box, to a glutamate completely inhibited export and processing of the fusion protein. The same mutation of R(-33), located outside the consensus box, had almost no effect. The data indicate a specific role for the consensus box sequence in the export mechanism for hydrogenase.

Amino Acid Sequence↗

PubMed1990

A new Drosophila homeo box gene is expressed in mesodermal precursor cells of distinct muscles during embryogenesis.

Several Drosophila homeo box genes have been shown to control cell fates in specific positions or cell groups of the embryo. Because the mechanisms involved in the pattern formation of complex internal organs, such as the musculature and the nervous system, are still largely unknown, we sought to identify and analyze new homeo box genes specifically expressed in these tissues. Here, the molecular analysis and expression pattern of one such gene, containing both a homeo box and a PRD repeat, is described. This gene, designated S59, is expressed in a small number of segmentally repeated mesodermal cells approximately 2 hr postgastrulation. Gradually, four groups of S59-expressing mesodermal cells appear in each abdominal hemisegment, each one giving rise to a particular somatic muscle after fusion with surrounding myoblasts. Thus, individual precursors for particular muscles, which we call "founder cells," are specified relatively early during mesodermal development. The expression of a particular homeo box gene in these cells suggests that distinct programs of gene expression are active in subsets of mesodermal cells after germ band elongation, resulting in a specification of their developmental fates. In addition to the mesoderm, S59 is expressed in a subset of neuronal cells of the CNS and their precursors and also in cells of a small region of the midgut.

Amino Acid Sequence↗

PubMed2000

Polymer confinement in undulated membrane boxes and tubes

We consider quantum particle or Gaussian polymer confinement between two surfaces and in cylinders with sinusoidal undulations. In terms of the variational method, we show that the quantum-mechanical wave equations have lower ground-state energy in these geometries under long wavelength undulations, where bulges are formed and waves are localized in the bulges. It turns out correspondingly that Gaussian polymer chains in undulated boxes or tubes acquire higher entropy than in exactly flat or straight ones. These phenomena are explained by the uncertainty principle for quantum particles, and by a polymer confinement rule for Gaussian polymers. If membrane boxes or tubes are flexible, polymer-induced undulation instability is suggested. We find that the wavelength of undulations at the threshold of instability for a membrane box is almost twice the distance between two walls of the box. Surprisingly, we find that the instability for tubes begins with a shorter wavelength compared to the "Rayleigh" area-minimizing instability.

Journal Article↗

PubMed2001

Liquid-solid phase transition of a system with two particles in a rectangular box.

We study the statistical properties of two hard spheres in a two-dimensional rectangular box. In this system, a relation similar to the van der Waals equation is obtained between the width of the box and the pressure working on the sidewalls. The autocorrelation function of each particle's position is calculated numerically. This calculation shows that, near the critical width, the time at which the correlation becomes zero gets longer as the height of the box increases. Moreover, fast and slow relaxation processes such as the alpha and beta relaxations in supercooled liquids are observed when the height of the box is sufficiently large. These relaxation processes are discussed with reference to the probability distribution of the relative positions of the two particles.

Journal Article↗

PubMed2000

Imaging the wave-function amplitudes in cleaved semiconductor quantum boxes

We have investigated the electronic structure of the conduction band states in InAs quantum boxes embedded in GaAs. Using cross-sectional scanning tunneling microscopy and spectroscopy, we report the direct observation of standing wave patterns in the boxes at room temperature. Electronic structure calculation of similar cleaved boxes allows the identification of the standing waves pattern as the probability density of the ground and first excited states. Their spatial distribution in the (001) plane is significantly affected by the strain relaxation due to the cleavage of the boxes.

Journal Article↗

PubMed2002

Ectopic expression of carpel-specific MADS box genes from lily and lisianthus causes similar homeotic conversion of sepal and petal in Arabidopsis.

Two MADS box genes, Lily MADS Box Gene 2 (LMADS2) and Eustoma grandiflorum MADS Box Gene 1 (EgMADS1), with an extensive similarity to the petunia (Petunia hybrida) FLORAL BINDING PROTEIN 7/11 and Arabidopsis AGL11, were characterized from the lily (Lilium longiflorum) and lisianthus (Eustoma grandiflorum). The expression of LMADS2 and EgMADS1 mRNA was restricted to the carpel and was absent in the other flower organs or vegetative leaves. LMADS2 mRNA was detected mainly in ovules and weakly in style tissues of the carpel, whereas EgMADS1 mRNA was only expressed in the ovules. Transgenic Arabidopsis plants ectopically expressing LMADS2 or EgMADS1 showed similar novel phenotypes resembling 35S::AGAMOUS plants by significantly reducing plant size, flowering early, and losing inflorescence indeterminacy. Ectopic expression of these two genes also generated similar ap2-like flowers by inducing homeotic conversion of the sepals into carpel-like structures in which stigmatic papillae and ovules were observed. In addition, the petals were converted into stamen-like structures in the second whorl of 35S::LMADS2 and 35S::EgMADS1 transgenic Arabidopsis. Our data indicated that LMADS2 and EgMADS1 are putative D functional MADS box genes in lily and lisianthus with a function similar to C functional genes once ectopically expressed in Arabidopsis.

Amino Acid Sequence↗

PubMed2006

Heterologous expression and molecular and cellular characterization of CaPUB1 encoding a hot pepper U-Box E3 ubiquitin ligase homolog.

The U-box motif is a conserved domain found in the diverse isoforms of E3 ubiquitin ligase in eukaryotes. From water-stressed hot pepper (Capsicum annuum L. cv Pukang) plants, we isolated C. annuum putative U-box protein 1 (CaPUB1), which encodes a protein containing a single U-box motif in its N-terminal region. In vitro ubiquitination and site-directed mutagenesis assays revealed that CaPUB1 possessed E3 ubiquitin ligase activity and that the U-box motif was indeed essential for its enzyme activity. RNA gel-blot analysis showed that CaPUB1 mRNA was induced rapidly by a broad spectrum of abiotic stresses, including drought, high salinity, cold temperature, and mechanical wounding, but not in response to ethylene, abscisic acid, or a bacterial pathogen, suggesting its role in the early events in the abiotic-related defense response. Because transgenic work was extremely difficult in hot pepper, in this study we overexpressed CaPUB1 in Arabidopsis (Arabidopsis thaliana) to provide cellular information on the function of this gene in the development and plant responses to abiotic stresses. Transgenic Arabidopsis plants that constitutively expressed the CaPUB1 gene under the control of the cauliflower mosaic virus 35S promoter had markedly longer hypocotyls and roots and grew more rapidly than the wild type, leading to an early bolting phenotype. Microscopic analysis showed that 35S::CaPUB1 roots had increased numbers of small-sized cells, resulting in disordered, highly populated cell layers in the cortex, endodermis, and stele. In addition, CaPUB1-overexpressing plants displayed increased sensitivity to water stress and mild salinity. These results indicate that CaPUB1 is functional in Arabidopsis cells, thereby effectively altering cell and tissue growth and also the response to abiotic stresses. Comparative proteomic analysis showed that the level of RPN6 protein, a non-ATPase subunit of the 26S proteasome complex, was significantly reduced in 35SCaPUB1 seedlings as compared to the wild type. Pull-down and ubiquitination assays demonstrated that RPN6 interacted physically with CaPUB1 and was ubiquitinated in a CaPUB1-dependent manner in vitro. Although the physiological function of CaPUB1 is not yet clear, there are several possibilities for its involvement in a subset of physiological responses to counteract dehydration and high-salinity stresses in transgenic Arabidopsis seedlings.

Amino Acid Motifs↗

PubMed1994

Two genes encoding GF14 (14-3-3) proteins in Zea mays. Structure, expression, and potential regulation by the G-box binding complex.

Two maize (Zea mays) genes, designated GRF1 and GRF2, have been isolated and characterized. The proteins encoded by these genes, called GF14 proteins, participate in protein/DNA complexes and show more than 60% identity with a highly conserved, widely distributed protein family, collectively referred to as 14-3-3 proteins. Members of the 14-3-3 protein family have been reported to activate Tyr and Trp hydroxylases, modulate protein kinase C activity, and activate ADP-ribosyltransferase. The mRNAs of the GRF genes are encoded by six exons interrupted by five introns. The transcriptional units of the GRF genes were found to be very similar, with complete conservation of the intron positions. In addition, the length and nucleotide sequences of the two genes' introns were highly conserved. The 5' flanking sequences of the two GRF genes were compared and regions of homology and divergence identified. This comparison revealed the presence of a conserved G-box element in the 5' flanking region of both genes. Electrophoretic mobility shift assays of maize protein extract with the GRF G-box indicates that GBF binds to this G-box site in the 5' up stream region of GRF. Antibody supershifts indicate that GF14 protein is associated with the G-box-binding complex that interacts with the GRF upstream region.

14-3-3 Proteins↗