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PubMed2004

BOX-pCR fingerprinting as a powerful tool to reveal synonymous names in the genus Streptomyces. Emended descriptions are proposed for the species Streptomyces cinereorectus, S. fradiae, S. tricolor, S. colombiensis, S. filamentosus, S. vinaceus and S. phaeopurpureus.

The type strains of 451 validly described Streptomyces species were screened using the BOX-PCR fingerprint technique. Of the different primers tested, the BOX primer yielded the most robust and reproducible band patterns. The majority of the species (350) had an unique pattern each. A total number of 30 clusters were delineated comprising species with nearly identical patterns. Four of these clusters grouped synonymous Streptomyces species known from previous studies. A high correlation exists between BOX-PCR fingerprinting and DNA-DNA homology data. In the present study, the latter was confirmed for 7 additional BOX clusters and led to the following emended descriptions: S. cochleatus is a subjective synonym of S. cinereorectus, S. roseoflavus of S. fradiae, S. roseodiastaticus of S. tricolor, S. roseosporus of S. filamentosus, S. distallicus of S. colombiensis, S. arabicus of S. vinaceus and S. phaeoviridis of S. phaeopurpureus. Although not yet confirmed by DNA-DNA pairing studies, rep-PCR data suggested the presence of at least 33 additional junior synonyms.

DNA Fingerprinting↗

PubMed1993

Influence of quantity of litter on nest box selection and nesting behaviour of domestic hens.

1. Twenty two hens (Gallus gallus domesticus) were given a free choice, for egg laying, between 4 nest boxes containing different quantities of wood shavings (litter): 3180 cm3 (large), 1060 cm3 (medium), 350 cm3 (small) and none (zero). 2. There was considerable individual variation in the quantity of litter selected for egg laying, but 17 hens laid 50% or more of their eggs in a single litter quantity. Hens selected the large quantity in preference to the small and zero. More searching, nest selection and nesting behaviours tended to be directed to the boxes with the largest quantities and also to the quantities that the hens finally selected for egg laying. 3. In a second experiment, the hens from experiment 1 which showed a preference for litter, and a further 22 naïve hens were given a choice of three nest boxes containing no litter and one containing one of 6 quantities. Over 30 trials there were 5 replicates using each of the three quantities used in experiment 1 plus 115 cm3, 40 cm3 and 10 cm3. 4. There was a wide range in the proportion of trials in which hens selected litter; for experienced hens the range was 0.43 to 0.97, but only two naïve hens were within this range, all others being between 0 and 0.43. 5. The pattern of choosing litter quantities was comparable but at different levels of preference in experienced and naïve hens and a contour map was derived enabling predictions to be made of the probability of a particular quantity being chosen. 6. The variability of individual responses suggest that no single type of nest box system can cater for the requirements of all hens.

Animals↗

PubMed2000

Development of a boxing dynamometer and its punch force discrimination efficacy.

The development of sport-specific dynamometers is an important step towards ecological validity in analysing athlete performance. Design limitations in previous punch-measuring devices have resulted in values which may not or cannot fully reflect the force and multidirectional components in a punch. In developing this boxing dynamometer, a triaxial force measurement system and a boxing manikin interface were combined. The repeatability and accuracy of the dynamomoter were assessed using simulated straight punches. Discrimination efficacy was assessed by comparison of the maximal punching force of seven elite, eight intermediate and eight novice boxers during simulated boxing, throwing straight punches. For the elite, intermediate and novice groups, respectively, the maximal straight punching forces (mean +/- s(mean)) were 4800 +/- 227 N, 3722 +/- 133 N and 2381 +/- 116 N for the rear hand, and 2847 +/- 225 N, 2283 +/- 126 N and 1604 +/- 97 N for the lead hand. For all groups, maximal forces were larger for the rear than the lead hand (P < 0.001). Maximal punching force was greater in the elite than the intermediate group, and greater in the intermediate than the novice group (P < 0.05). The boxing dynamometer discriminated effectively between punching performance at three standards of performance and between the punching force of the rear and lead hands.

Biomechanical Phenomena↗

PubMed1997

The SRY cantilever motif discriminates between sequence- and structure-specific DNA recognition: alanine mutagenesis of an HMG box.

The high-mobility-group (HMG) box defines a DNA-bending motif conserved among architectural transcription factors. A "hydrophobic wedge" at the protein surface provides a mechanism of DNA bending: disruption of base stacking by insertion of a sidechain "cantilever." First described in the mammalian testis-determining factor SRY, the cantilever motif consists of adjacent aromatic and nonpolar sidechains at the crux of the HMG box (residues 12 and 13). Here, the role of these side chains in DNA recognition is investigated by alanine mutagenesis. F12A and I13A substitutions in the SRY HMG box each permit native folding and thermal stability (as monitored by circular dichroism and 1H-NMR) but eliminate sequence-specific DNA-binding activity (as detected by gel-mobility shift). On binding to the sharp angles of a four-way DNA junction (4WJ), however, the substitutions each promote formation of a high-molecular-weight aggregate, presumably by DNA-dependent oligomerization. The substitutions have opposite effects on initial binding to the 4WJ: whereas such binding is attenuated ten-fold by F12A, it is enhanced by I13A. A foreshortened "alanine cantilever", not observed among specific HMG boxes, occurs in a non-specific domain (HMG-1A) and may enhance architecture-selective DNA recognition.

Alanine↗

PubMed1998

Box Handling in the Loading and Unloading of Vans.

The handling of 2,306 boxes being loaded or unloaded from vans onto or from 4-wheeled trolleys by 31 handlers in a warehouse were characterized. Handling was videotaped and characterized through an analysis grid completed by three trained observers. The following execution parameters were observed: nature of the exertion applied by the upper limbs, plane and direction of the exertion, resulting displacement of the box, grip, use of the lower limbs and the back. Results show that execution parameters used by handlers vary considerably from those usually recommended or studied. For example, symmetric grips were rarely used (4%). The grip was modified during the handling of half the boxes. Significant knee flexion was rarely observed (3% of exertions). Each box was moved by applying an average of 3.5 different exertions. Exertions were mostly applied in a plane parallel to the shoulders; they were rarely executed in a strict sagittal plane (11%). The implication of these observations are discussed.

Journal Article↗

PubMed2005

Phosphorous sorption by Filtralite P--small scale box experiment.

Phosphorus (P) sorption of light weight aggregate, Filtralite P has been examined through a box experiment which imitates a horizontal subsurface flow wetland system. The results showed that after the P breakthrough, the outlet P concentration increased with time according to the amount of P applied. Small scale boxes with a high inlet P concentration (15 ppm) and high loading rate (5-2.5 L d(-1)) reached 90% saturation level relatively quickly (after about 150 days of operation), while the boxes with low hydraulic loading rate (1.25 L day(-1)) were 70-90% saturated after 18 months of operation. The total P removal was dependent on pH, Ca, and the inlet P concentrations, but was independent of the hydraulic loading rate. Extraction of total P from the saturated filter material showed that the sorbed P accumulated within the inlet section of the box and decreased gradually towards the outlet as well as towards the bottom layer. Even after large amounts of Ca had leached out of the system, Filtralite P still had a very high P removal capacity. After resting periods the P sorption capacity of the material was regenerated, the P concentration in the effluent decreased by 22-53%.

Adsorption↗

PubMed1992

Centromere protein B assembles human centromeric alpha-satellite DNA at the 17-bp sequence, CENP-B box.

We purified 15,000-fold from HeLa cell nuclear extract the centromere antigen that reacts specifically with the 17-bp sequence, designated previously as CENP-B box, in human centromeric alpha-satellite (alphoid) DNA by a two-step procedure including an oligonucleotide affinity column. The purified protein was identified as the centromere protein B (CENP-B) by its mobility on SDS-PAGE (80 kD), and reactivities to a monoclonal antibody raised to CENP-B (bacterial fusion protein) and to anticentromere sera from patients with autoimmune diseases. Direct binding by CENP-B of the CENP-B box sequence in the alphoid DNA has been proved using the purified CENP-B by DNA mobility-shift assay, Southwestern blotting, and DNase I protection analysis. The binding constant of the antigen to the CENP-B box sequence is 6 x 10(8) M-1. DNA mobility-shift assays indicated that the major complex formed between the CENP-B and the DNA contains two DNA molecules, suggesting the importance of the CENP-B/CENP-B box interaction in organization of higher ordered chromatin structures in the centromere and/or kinetochore. Location of DNA binding and dimerization domains in CENP-B was discussed based on the DNA mobility-shift assays performed with a protein fraction containing intact and partial cleavage products of CENP-B.

Autoantigens↗

PubMed1993

Repression of major histocompatibility complex IA expression by glucocorticoids: the glucocorticoid receptor inhibits the DNA binding of the X box DNA binding protein.

Glucocorticoids are effective repressors of major histocompatibility complex (MHC) class II gene expression. The repression occurs in B cells, which constitutively express MHC class II, as well as in macrophages, which only express MHC class II after the cells are treated with interferon gamma. For the MHC class II gene IA beta, this negative regulation has been linked to the X box DNA sequence, located with the IA beta promoter. The addition of the glucocorticoid receptor was shown to inhibit the DNA binding of the X box DNA binding protein to the X box. The DNA binding of two other DNA binding proteins that recognize elements within this promoter was unaffected by the addition of glucocorticoid receptor. It is likely that the repression of IA beta gene expression by glucocorticoids occurs because the X box DNA binding protein is prevented from binding to the DNA and activating transcription.

Animals↗

PubMed2002

A newly identified chromosomal microdeletion and an N-box mutation of the AChR epsilon gene cause a congenital myasthenic syndrome.

Congenital myasthenic syndromes (CMSs) are frequently caused by mutations of the coding region of the acetylcholine receptor epsilon subunit (AChRepsilon) gene leading to a reduced expression of the acetylcholine receptor (AChR) at the postsynaptic membrane. Two recent observations have linked two different N-box mutations of the human AChRepsilon promoter to a clinical CMS phenotype. N-boxes are regulatory sequence elements of mammalian promoters that confer synapse-specific expression of several genes, including the AChR subunit genes. Here, we report on a novel point mutation (epsilon-154G-->A) in the N-box of the AChRepsilon promoter in a German CMS pedigree. Semiquantitative analysis of AChRepsilon mRNA levels in the patient's muscle indicated significantly impaired AChRepsilon expression. We provide additional evidence of a pathogenic role for this mutation using the mutated promoter (epsilon-154G-->A) driving a heterologous gene (luciferase) in rat skeletal muscle. We show that agrin-induced gene expression is significantly reduced by the N-box mutant (mt) compared with the wild-type (wt) promoter. Refined haplotype analysis and direct sequencing revealed maternal inheritance of the mutant AChRepsilon promoter (epsilon-154G-->A) together with paternal inheritance of a chromosomal microdeletion (Delta1290 bp) encompassing the promoter and the first two exons of the AChRepsilon gene in the index patient. In conclusion, we provide genetic and functional evidence that a mutation of the AChRepsilon subunit promoter (epsilon-154G-->A) causes CMS due to the reduction of gene expression in skeletal muscle. Moreover, this is the first report of a chromosomal microdeletion affecting an AChR gene. This type of mutation may be missed in standard screening techniques of CMS patients.

Adult↗

PubMed1997

Molecular characterization of the B-box protein-protein interaction motif of the ETS-domain transcription factor Elk-1.

The ternary complex factor (TCF) subfamily of ETS-domain transcription factors form ternary complexes with the serum response factor (SRF) and the c-fos SRE. Extracellular signals are relayed via MAP kinase signal transduction pathways through the TCF component of the ternary complex. Protein-protein interactions between TCFs and SRF play an essential role in formation of this ternary complex. A 30 amino acid sequence encompassing the TCF B-box is sufficient to mediate interactions with SRF. In this study we have identified amino acids which are critical for this interaction and derived a molecular model of the SRF binding interface. Alanine scanning of the Elk-1 B-box reveals five predominantly hydrophobic residues which are essential for binding to SRF and for ternary complex formation in vitro and in vivo. These amino acids are predicted to lie on one face of an alpha-helix. Peptides encompassing the B-box retain biological activity and have helix-forming propensity. alpha-Helix and ternary complex formation is disrupted by the introduction of helix-breaking proline residues. Our results are consistent with a model in which the Elk-1 B-box forms an inducible alpha-helix which presents a hydrophobic face for interaction with SRF. We discuss the wider applicability of our results to similar short protein-protein interaction motifs found in other transcription factors.

3T3 Cells↗

PubMed1995

Molecular evolution of flower development: diversification of the plant MADS-box regulatory gene family.

Floral homeotic genes that control the specification of meristem and organ identity in developing flowers have been isolated from both Arabidopsis thaliana and Antirrhinum majus. Most of these genes belong to a large family of regulatory genes and possess a characteristic DNA binding domain known as the MADS-box. Members of this gene family display primarily floral-specific expression and are homologous to transcription factors found in several animal and fungal species. Molecular evolutionary analyses reveal that there are appreciable differences in the substitution rates between different domains of these plant MADS-box genes. Phylogenetic analyses also demonstrate that members of the plant MADS-box gene family are organized into several distinct gene groups: the AGAMOUS, APETALA3/PISTILLATA and APETALA1/AGL9 groups. The shared evolutionary history of members of a gene group appear to reflect the distinct functional roles these MADS-box genes play in flower development. Molecular evolutionary analyses also suggest that these different gene groups were established in a relatively short span of evolutionary time and that the various floral homeotic loci originated even before the appearance of the flowering plants.

Amino Acid Sequence↗

PubMed1994

Distribution of CENP-B boxes reflected in CREST centromere antigenic sites on long-range alpha-satellite DNA arrays of human chromosome 21.

The long-range organization of alphoid DNA arrays of human chromosome 21 was investigated using a mouse-human somatic cell hybrid. Two distinct long alphoid DNA arrays, the loci alpha 21-I and alpha 21-II, were identified in the centromere region of human chromosome 21. The alpha 21-I locus, composed of an array of 11 monomer repeat units (the 11 mer), was estimated to have a total length of 1.3 Mbp. CENP-B boxes, the binding sites of the centromere protein B (CENP-B), appeared in every other monomer unit in the 11 mer except for one place where two monomer units were repeated without any CENP-B box. The other locus, alpha 21-II, was found to be composed of alphoid subfamilies with low homology to the components of alpha 21-I locus. Five different alphoid clones presenting 32 monomer units in total were isolated from the alpha 21-II locus. Sequences of these monomer units diverged between 71-89% and no unit containing a CENP-B box was found. By analysis using two color FISH, the alpha 21-I was localized to the primary constriction, whereas the alpha 21-II site was located slightly to the short arm side. Furthermore, a combination of FISH and immunofluorescent staining indicated that the alpha 21-I site was co-localized and overlapped with the CREST centromere antigenic site on mitotic chromosomes and in interphase nuclei, while alpha 21-II was distributed broadly. Our data suggest that the locus alpha 21-I containing regularly spaced CENP-B boxes at high-frequency and the assembly site of the centromere antigens may be involved in common centromere function in both human and mouse cells.

Animals↗

PubMed2000

Birds of a feather: using a rotational box plot to assess ascertainment bias.

BACKGROUND: Comparability of study participants with non-participants is customarily assessed by contrasting the distributions of sociodemographic characteristics. Such comparisons do not necessarily provide insight into whether or not participants of a given subgroup are similar to non-participants of the same subgroup. A geographical information system (GIS) may provide such insight by visually displaying the spatial distributions of participants and non-participants. In a previously reported study of heterosexuals at elevated risk for human immunodeficiency virus (HIV), traditional methods suggested distributional differences in the demographic characteristics of participants and non-participants. METHODS: Based on residential address co-ordinates for each subgroup member, we used the subgroup's centroid as the origin and constructed a 360 degrees series of overlapping box plots of the distance of subgroups members to the origin, thereby producing closed polygons for each of the box plot demarcators. RESULTS: These rotational box plots revealed similar geographical distributions for most participant and non-participant subgroups, with the exception of African-American men and women. CONCLUSIONS: Observed differences resulted in part from the study design, and provided some insight into sampling problems encountered in social network studies. Based on Tobler's supposition that 'nearby things tend to be alike', the rotational box plot is a useful additional tool for investigating sample bias.

Adult↗

PubMed2003

Mutagenesis analysis of the interaction between the dorsal rel homology domain and HMG boxes of DSP1 protein.

DSP1 is an HMG-like protein of Drosophila melanogaster consisting of 386 amino acids with two HMG domains at the C-terminal end. It was shown to interact with Dorsal protein through the HMG domains and to enhance its DNA binding. Each HMG domain consists of approximately 80 amino acid residues, forming three alpha helices folded into an L-shaped structure. We have compared the interaction of various truncated and mutated forms of DSP1 with the dorsal Rel homology domain (RHD). In particular, we have mutated the conserved tryptophan residue 212 or 302 in A or B boxes or the lysine-rich region ((253)KKRK(256)) of the A/B linker. Analysis by circular dichroism revealed that the protein tertiary structure is affected in these mutants. However, these mutations do not abolish the DSP1 binding to Dorsal, except if the two HMG boxes are altered, i.e., in a double mutant or in mutant isolated domain. Finally, studies on the enhancement of Dorsal DNA binding by DSP1 revealed that the DNA affinity is maximum in the presence of wild-type DSP1, is dramatically reduced when box A is altered, and is completely abolished when box B is altered.

Amino Acid Sequence↗

PubMed2005

In vivo imaging of MADS-box transcription factor interactions.

MADS-box transcription factors are major regulators of development in flowering plants. The factors act in a combinatorial manner, either as homo- or heterodimers, and they control floral organ formation and identity and many other developmental processes through a complex network of protein-protein and protein-DNA interactions. Despite the fact that many studies have been carried out to elucidate MADS-box protein dimerization by yeast systems, very little information is available on the behaviour of these molecules in planta. Here, evidence for specific interactions between the petunia MADS-box proteins FBP2, FBP11, and FBP24 is provided in vivo. The dimers identified in yeast for the ovule-specific FBP24 protein have been confirmed in living plant cells by means of fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy and, in addition, some of the most likely, less stable homo- and heterodimers were identified. This in vivo approach revealed that particular dimers could only be detected in specific sub-nuclear domains. In addition, evidence for the in planta assembly of these ovule-specific MADS-box transcription factors into higher-order complexes is provided.

Binding, Competitive↗

PubMed2006

Molecular and phylogenetic analyses of the MADS-box gene family in tomato.

MIKCc-type MADS-box genes encode key transcriptional regulators of a variety of developmental processes in Arabidopsis thaliana. However, there has been relatively little effort to systematically carry out comparative genomic or functional analyses of these genes across flowering plants. Here we describe a strategy to identify members of the MIKCc-type MADS-box gene family from any angiosperm species of interest. Using this approach, we have identified 24 MIKCc-type MADS-box genes in tomato, including 17 that have not previously been characterized. Using these sequences, we have performed phylogenetic analyses that indicate that there have been a number of gene duplication and loss events in tomato relative to Arabidopsis. We also describe the expression domains of these genes and compare these results with their cognates in Arabidopsis. These analyses demonstrate the utility of this approach for characterizing a large number of MIKCc-type MADS-box genes from any flowering plant species of interest and provide a framework for evolutionary comparisons of this important gene family across angiosperms.

Arabidopsis↗

PubMed1986

The effect of changing the distance between the TATA-box and cap site by up to three base pairs on the selection of the transcriptional start site of a cloned eukaryotic gene in vitro and in vivo.

We have studied how small changes in the distance between the TATA-box and cap site affect transcription of a eukaryotic gene in vitro and in vivo. The trout protamine gene TPG-3 [Gregory et al. (1982) Nucl. Acids Res. 10, 7581-7592] is a good model for such a study as it has (i) a consensus TATA-box 32 base pairs (bp) upstream from an A-residue which is the natural cap site (designated +1) (ii) two further A-residues at -5 and +5, providing alternative transcriptional start sites which are in significantly different sequence environments and (iii) a unique AvaII restriction site immediately downstream from the TATA-box which is ideal for the insertion or deletion of up to 3bp. Transcripts of the wild type and mutant genes were generated in vitro using a HeLa whole cell extract or 'in vivo' by transient expression following their transfection into HeLa cells. These 'spacer' mutations did not affect the efficiency of transcription of the gene in vitro but they did affect the selection of transcriptional start site both in vitro and 'in vivo'. Analysis of 5'-ends by S1-mapping and primer extension showed that the A-residue(s) selected are those which, by insertion or deletion, come to lie on the same face of the DNA double helix as the TATA-box, although the DNA sequence in the immediate vicinity of the potential start sites influences their utilisation. Comparison of the TPG-3 wild type transcripts in these experimental systems with natural mRNA suggests that cap site selection is more stringent in the developing trout testis.

Animals↗

PubMed1988

An erythroid specific nuclear factor binding to the proximal CACCC box of the beta-globin gene promoter.

We have used the gel retardation and DNAase I assays to investigate the binding of nuclear proteins to the human beta-globin promoter. Upon incubation with beta-globin promoter fragments containing the duplicated CACCC boxes, nuclear proteins from human erythroid cells generate complexes yielding four retarded bands in acrylamide gels; the three slowest bands are common to both erythroid and non erythroid cells. The fast band is present only in K562 erythroleukemic cells induced to differentiation and hemoglobin accumulation and in fetal and adult erythroblasts, but absent in uninduced K562 cells. Binding occurs on a short DNA region including the proximal CACCC box, and is not significantly competed by excess gamma-globin fragments containing the CACCC box; the CACCC box appears to be essential for this binding, as shown by the failure of a fragment containing a natural beta-thalassemic mutation (-87, C----G) to bind significantly to nuclear factors. These data suggest that the erythroid specific CACCC binding factor might play a role in the developmental activation of beta-globin transcription.

Base Sequence↗