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PubMed1994

Box I and II motif from myelin basic protein gene promoter binds to nuclear proteins from rodent brain.

The box I and II motif located within the promoter of the myelin basic protein gene contains a simian virus 40 T-antigen-binding site, a MyoD/E2a-binding site, and a glucocorticoid receptor-binding site. We have found proteins within nuclear extracts from adult mouse brain, rat embryonic cerebral cortex in culture, and a mouse oligodendrocyte-like cell line that bind to a 32P-labeled synthetic DNA fragment containing the sequences of the box I and II motif. Three major complexes (A, B, and C) were seen in gel-shift assays. Only complexes A and B were competed out by the unlabeled box I and II fragment or by another synthetic DNA fragment that also contains sequences similar to a glucocorticoid receptor-binding site. Therefore, complexes A and B were thought to be specific. The expression pattern of the proteins responsible for the formation of these complexes was also assessed during development in mouse brain.

Animals↗

PubMed2000

Paradoxical effect of inserting, in Enterococcus faecalis penicillin-binding protein 5, an amino acid box responsible for low affinity for penicillin in Enterococcus faecium.

Penicillin-binding proteins 5 (PBP5s) of enterococci are structurally and immunologically related proteins that are characterized by their low affinity for penicillin. For this reason, they are mainly involved in penicillin resistance, due essentially to their ability to take over the function of all other PBPs already bound and inhibited by the beta-lactam. It has been demonstrated that penicillin resistance in enterococci is acquired either by overproduction of PBP5 or by the presence of specific amino acid sequences in the protein that further decrease the affinity for penicillin. In particular, a specific amino acid box (ANNGA) previously identified in Enterococcus faecium is responsible for the high penicillin resistance displayed by this species. Here, we describe the insertion of the PBP5 amino acid box ANNGA in Enterococcus faecalis, an enterococcal species usually more sensitive to penicillin, by site-directed mutagenesis. Mutagenized PBP5 was re-introduced into a pbp5 mutant of E. faecalis obtained by insertion of transposon Tn916. Data indicate that this amino acid box brings about no reduction in penicillin sensitivity in the recipient E. faecalis strain, but, paradoxically, dramatically lowers the penicillin minimal inhibitory concentration caused by the native PBP5. We deduce that, although enterococcal PBP5s are a family of closely related proteins as far as biological function is concerned, differences exist in their three-dimensional structure that affect penicillin affinity.

Amino Acid Sequence↗

PubMedDate not supplied

Evolution of MADS-box gene induction by FLO/LFY genes.

Some MADS-box genes function as floral homeotic genes. The Arabidopsis LFY gene is a positive regulator of floral homeotic genes, and homologs of the FLO/LFY gene family in other angiosperms and gymnosperms are likely to have a similar function. To investigate the origin of the floral homeotic gene regulatory cascade involving the FLO/LFY gene, FLO/LFY homologs were cloned from a leptosporangiate fern (Ceratopteris richardii), two eusporangiate ferns (Angiopteris lygodiifolia and Botrychium multifidum var. robustum), three fern allies (Psilotum nudum, Equisetum arvense, and Isoetes asiatica), and a moss (Physcomitrella patens). The FLO/LFY gene phylogenetic tree indicates that both duplication and loss of FLO/LFY homologs occurred during the course of vascular plant evolution. The expression patterns of the Ceratopteris LFY genes (CrLFY1 and 2) were assessed. CrLFY1 expression was prominent in tissues including shoot tips and circinate reproductive leaves, but very weak in other tissues examined. Expression of CrLFY2 was also prominent in tissues, including shoot tips and circinate reproductive leaves. These patterns of expression are dissimilar to that of any Ceratopteris MADS-box gene previously reported, suggesting that the induction of MADS-box genes by FLO/LFY is not established at the stage of ferns.

Arabidopsis↗

PubMed2002

Enhanced expression of tandem multimers of the antimicrobial peptide buforin II in Escherichia coli by the DEAD-box protein and trxB mutant.

The tandem multimeric expression of various peptides has been explored by many researchers. However, expression levels have usually not been proportional to the degree of multimerization. To increase the expression level in Escherichia coli of tandem multimers of a cationic antimicrobial peptide, buforin II, fused to an anionic peptide, we studied the effect of the DEAD-box protein and the trxB mutant on the expression of tandem multimers. An expression vector with a tac promoter was more effective in directing multimeric expression than one with a T7 promoter. The expression level of large multimers was substantially increased with the tac promoter, possibly through stabilization of long transcripts by synchronization of transcription and translation. Coexpression of the DEAD-box protein, an RNA-binding protein, with the T7 expression system increased the expression level of multimers, especially large multimers, due to protection of the long RNA transcripts. In addition, the use of the trxB mutant also enhanced the expression level of tandem multimers, which contain two cysteine residues at both ends of the monomeric unit. It seems that disulfide bonds formed in the multimers in the trxB mutant might help efficient charge neutralization for inclusion body formation of the multimers, resulting in enhancement of expression. Our results show that the expression of multimers can be improved through the stabilization of the long transcripts by the DEAD-box protein or the expression, under an oxidizing environment, of the trxB mutant in which covalent cross-links through disulfide bonds facilitate inclusion body formation of the multimeric fusion peptide.

Anti-Bacterial Agents↗

PubMed1998

Gender comparison of peak oxygen uptake: repetitive box lifting versus treadmill running.

The gender differences in peak oxygen uptake (VO2peak) for various modes of exercise have been examined previously; however, no direct gender comparisons have been made during repetitive lifting (RL). In the present study the VO2peak between RL and treadmill running (TR) was compared between 20 men [mean (SD) age, height, body mass and body fat: 21 (3) years, 1.79 (0.06) m, 81 (9) kg, 19 (6)%, respectively] and 20 women [mean (SD) age, height, body mass and body fat: 21 (3) years, 1.63 (0.05) m, 60 (7) kg, 27 (6)%, respectively]. VO2peak (l x min[-1]), defined as the highest value obtained during exercise to volitional fatigue, was determined using discontinuous protocols with treadmill grade or box mass incremented to increase exercise intensity. For RL VO2peak, a pneumatically driven shelf was used to lower a loaded box to the floor, and subjects then lifted the box, at a rate of 15 lifts x min(-1). VO2peak (l x min(-1) and ml x kg(-1) x min[-1]) and minute ventilation (VE, l x min[-1]) were determined using an on-line gas analysis system. A two-way repeated measures analysis of variance revealed significant gender effects, with men having higher values for VO2peak (l x min(-1) and ml x kg(-1) x min[-1]) and VE, but women having higher values of the ventilatory equivalent for oxygen (VE/VO2). There were also mode of exercise effects, with TR values being higher for VO2peak (l x min(-1) and ml x kg(-1) x min[-1]) and VE and an interaction effect for VO2peak (l x min(-1) and ml x kg(-1) x min[-1]) and VE/VO2. The women obtained a greater percentage (approximately 84%) of their TR VO2peak during RL than did the men (approximately 79%). There was a marginal tendency for women to decrease and men to increase their VE/VO2 when comparing TR with RL. The magnitude of the gender differences between the two exercise modalities appeared to be similar for heart rate, VE and R, but differed for VO2peak (l x min(-1) and ml x kg(-1) x min[-1]). Lifting to an absolute height (1.32 m for the RL protocol) may present a different physical challenge to men and women with respect to the degree of involvement of the muscle groups used during lifting and ventilation.

Adult↗

PubMed2003

Pistillody is caused by alterations to the class-B MADS-box gene expression pattern in alloplasmic wheats.

Class-B floral homeotic genes are involved in specifying petal and stamen identity during flower development in plant species. Homeotic transformation of stamens into pistil-like structures (called pistillody) has been observed in alloplasmic lines of bread wheat ( Triticum aestivum L.) having the cytoplasm of a wild relative species, Aegilops crassa Boiss. To obtain information about the molecular mechanism underlying pistillody induction, we isolated two PISTILLATA ( PI)-type class-B MADS-box genes, WPI1 ( wheat PI STILLATA # 1) and WPI2, from wheat. Phylogenetic reconstruction indicated that WPI1 is orthologous to OsMADS4 and that WPI2 is probably an ortholog of OsMADS2. Both OsMADS4 and OsMADS2 genes were suggested to be PI orthologs in rice, and the function of OsMADS4 as a class-B gene was proven by the transgenic study. An in situ hybridization study demonstrated that the WPI1 gene is expressed in primordia of lodicules and stamens in developing florets in wheat. In the alloplasmic wheat line exhibiting pistillody, the WPI1 transcripts were not detected in the primordia of pistil-like stamens, whereas WPI1 was expressed in the lodicules. The wheat APETALA3 ( AP3)-type class-B MADS-box gene WAP3 ( wheat AP3) /TaMADS#82 showed an expression pattern similar to that of WPI1. These results suggest that pistillody in alloplasmic wheats is caused by alterations to the expression pattern of class-B MADS-box genes.

Amino Acid Sequence↗

PubMed2001

Isolation of a MADS-box gene (ERAF17) and correlation of its expression with the induction of formation of female flowers by ethylene in cucumber plants (Cucumis sativus L.).

Ethylene regulates sex expression in cucumber (Cucumis sativus L.) plants. When the apices of monoecious cucumber seedlings (cv. Shimoshirazu-jibai) were treated with the ethylene-releasing compound, ethephon, female flowers were induced at the nodes. To clarify the action of ethylene in the regulation of sex expression, we attempted to isolate genes whose expression changed during induction of the formation of female flowers at the apices of these cucumber plants upon treatment with ethephon. Using the differential-display method, we identified 20 clones (#1 to #20) that reflected differences in the accumulation of transcripts in apices treated or not treated with ethephon. Sequence analysis of cDNA fragments revealed that the cDNA #17 had the sequence of a MADS-box gene. We isolated the full-length cDNA and showed that it included both a MADS box and a K box, and the corresponding gene was designated ERAF17. We examined the expression of ERAF17 in the apices of cv. Shimoshirazu-jibai and in those of a gynoecious cultivar (Rensei). In these cultivars, the timing and levels of expression of the ERAF17 transcript were correlated with the development of female flowers. Induction of the synthesis of the ERAF17 transcript by ethephon occurred within 4 h of the start of treatment and continued for 4 days at least. Expression of ERAF17 at apices was localized in the floral buds of the gynoecious cultivar, and expression was maintained in female flowers thorough their development. Our results suggest that the induction of the formation of female flowers by ethylene might be regulated by the expression of ERAF17 in floral buds at the apices of cucumber plants and that expression of this gene might also be involved in the development of female flowers.

Amino Acid Sequence↗

PubMed1999

DEAD-box protein HEL64 from Trypanosoma brucei: subcellular localization and gene knockout analysis.

RNA helicases are molecules that play a central role in the control of ribonucleic acid metabolism. Only two putative RNA helicase genes of the DEAD-box family have been identified in the protozoan parasite Trypanosoma brucei brucei. One of these genes is HEL64, a single-copy gene of unknown function. In this study we conducted targeted gene-disruption experiments of the HEL64 locus with the aim of identifying a phenotype that would suggest a function for the encoded protein. It is likely that HEL64 is an essential gene in insect-stage trypanosomes, since all attempts to create a HEL64 double-allele knockout cell line failed. Instead, we obtained a mutant derived from an unusual recombination event that nonetheless contained a functional copy of the gene. One allele of HEL64 is sufficient for the survival of the parasite. Single-allele knockout mutants showed no gross change in cell morphology and multiplied with a cell-doubling time identical to that of wild-type trypanosomes. Though HEL64 has high sequence homology with the nuclear DEAD-box protein p68, it is localized in the cytosol of trypanosomes and, thus, cannot be a homologue of p68 as previously suggested. Since the protein did not cross-hybridize with an anti-eIF-4A antibody, we excluded the possibility that HEL64 might be a homologue of the translation initiation factor eIF-4A. Although the function of HEL64 remains unknown, the present data indicate that the encoded DEAD-box protein plays an important role during the insect life-cycle stage of the parasite.

Alleles↗

PubMed2002

The tobacco bZIP transcription factor BZI-1 binds to G-box elements in the promoters of phenylpropanoid pathway genes in vitro, but it is not involved in their regulation in vivo.

Screening of a tobacco (Nicotiana tabacum) cDNA library resulted in the isolation of a clone encoding the bZIP transcription factor BZI-1. With respect to amino acid sequence, conservation of protein domains, genomic exon-intron structure and expression pattern, BZI-1 is closely related to CPRF2, OHP1/2, BLZ1 and REB, a group of bZIP proteins which have been described in a number of dicot and monocot species. BZI-1 exhibits the characteristics of a transcription factor. It binds to G-box and C-box cis-elements in vitro, it is localised in the nucleus, and the N-terminal region of BZI-1 functions as an activation domain in both yeast and plant cells. Since BZI-1-related transcription factors have been isolated from dicots by in vitro binding to G-box elements in the chalcone synthase ( CHS) promoter, it has been suggested that phenylpropanoid pathway genes, such as CHS and PAL (phenylalanine ammonia-lyase), are targets of these proteins in vivo. However, after infection with Pseudomonas syringae or Tobacco Mosaic Virus, no changes in pathogen-induced PAL expression were observed in transgenic plants expressing increased levels of BZI-1 or a dominant negative form of the protein, BZI-1-DeltaN. In contrast to the tissue-specific expression of CHS and PAL, BZI-1 was found to be ubiquitously expressed in tobacco plants. Furthermore, no changes in the tissue-specific expression of PAL or CHS were observed in plants that were transgenic for BZI-1-DeltaN. Expression of a VP16-BZI-1 fusion protein would be expected to result in constitutive activation of the BZI-1 target genes. However, tetracycline-dependent expression of a VP16-BZI-1 protein in tobacco plants did not result in activation of CHS or PAL. On the basis of these data, we conclude that the phenylpropanoid pathway genes analysed are not targets of BZI-1 in vivo. Thus, the pattern of in vitro DNA binding of transcription factors need not always reflect their in vivo function.

Amino Acid Sequence↗

PubMed2003

Analysis of the petunia MADS-box transcription factor family.

Transcription factors are key regulators of plant development. One of the major groups of transcription factors is the MADS-box family, of which at least 80 members are encoded in the Arabidopsis genome. In this study, 23 members of the petunia MADS-box transcription factor family were investigated by Northern hybridisation, phylogenetic and yeast two-hybrid analyses. Many of the genes characterised appeared to have one or more close relatives that shared similar expression patterns. Comparison of the binding interactions of these proteins revealed that some show similar interaction patterns, and hence are likely to be functionally redundant. From an evolutionary point of view, their coding genes are probably derived from a recent duplication event. Furthermore, protein-protein interaction patterns, in combination with expression patterns and phylogenetic classification, appear to offer good criteria for the identification of functional homologues. Based on comparison of such data between petunia and Arabidopsis, functions can be predicted for several MADS-box transcription factors in both species.

Arabidopsis↗

PubMed1997

MADS box genes expressed in developing inflorescences of rice and sorghum.

With the aim of elucidating the complex genetic system controlling flower morphogenesis in cereals, we have characterized two rice and two sorghum MADS box genes isolated from cDNA libraries made from developing inflorescences. The rice clones OsMADS24 and OsMADS45, which share high homology with the Arabidopsis AGL2 and AGL4 MADS box genes, are expressed in the floral meristem, in all the primordia, and in mature floral organs. High expression levels have also been found in developing kernels. The sorghum clone SbMADS1 is also homologous to AGL2 and AGL4: expression analysis and mapping data suggest that it is the ortholog of OsMADS24. The pattern of expression of SbMADS2, the other sorghum MADS box gene, suggests that it may play a role as a meristem identity gene, as does AP1 in Arabidopsis, to which it shows considerable homology. The four genes have been mapped on a rice RFLP genetic map: the results are discussed in terms of synteny among cereals.

Amino Acid Sequence↗

PubMed2003

Seed dispersal by the Florida box turtle (Terrapene carolina bauri) in pine rockland forests of the lower Florida Keys, United States.

Seed dispersal by animals is one of the most important plant-animal mutualisms, but saurochory, the dispersal of seeds by reptiles, has received little attention. We investigated the role of the Florida box turtle (Terrapene carolina bauri) as a seed dispersal agent in pine rockland forests of the lower Florida Keys and examined the effect of turtle digestion on seed germination. We obtained seeds of 11 species with fleshy fruits and 2 species with non-fleshy fruits (a grass and legume) from the feces of 145 box turtles collected on Key Deer National Wildlife Refuge from 1999 to 2000. We planted the seeds of nine species and germination percentage (percentage of seeds that germinated during the experiment) varied from 10% to 80%. Comparative germination experiments were conducted with Thrinax morrissii, Serenoa repens, and Byrsonima lucida. We compared the germination percentage and germination rate (number of days from planting to seedling emergence) of seeds from three treatments (seeds recovered from feces, control seeds with pulp, and control seeds without pulp) and continued these experiments for up to 2 years. Passage through the box turtle digestive tract greatly enhanced the germination percentage and germination rate of S. repens, but decreased the germination percentage of B. lucida and T. morrissii, and decreased germination rate for T. morrissii. Subsequent destructive seed viability tests revealed that many ungerminated T. morrissii seeds remained viable, suggesting long-term seed dormancy may occur, even after passage through the turtle digestive system. In addition, the proportion of ungerminated seeds which remained viable was greater for seeds recovered from turtle feces than from control seeds with pulp. Furthermore, removal of fleshy pulp either manually or by the turtle digestive system may allow T. morrissii to escape insect predation.

Animals↗

PubMed2005

BOX-PCR is an adequate tool for typing Aeromonas spp.

PCR-based methods of fingerprinting take advantage of the presence of repetitive sequences that are interspersed throughout the genome of diverse bacterial species. They include the repetitive extragenic palindromic (REP) sequence, the enterobacterial repetitive intergenic consensus sequence (ERIC) and the 154-bp BOX element. The combination of the three methods is used for fine discrimination of strains and is designated as rep-polymerase chain reaction (PCR). REP-PCR and ERIC-PCR have been shown to be useful for typing Aeromonas strains. To our knowledge, rep-PCR fingerprinting method using the BOXA1R primer has never been tested on aeromonads. In this study, the BOX-PCR fingerprinting technique was evaluated for the discrimination of strains of some Aeromonas species. All strains were typeable and the majority showed unique banding patterns. Four strains from culture collections were used to investigate the reproducibility of the method. According to our results, BOX-PCR fingerprinting is applicable for typing of Aeromonas strains and can be considered as a useful complementary tool for epidemiological studies of members of this genus.

Aeromonas↗

PubMed2005

Translation enhancement by optimized downstream box sequences in Escherichia coli and Mycobacterium smegmatis.

The effect on translation of downstream box sequences optimized for binding to Mycobacterium smegmatis and Escherichia coli 16S rRNA in the absence of a Shine-Dalgarno (SD) region was investigated. The relative translational efficiency of each construct in either M. smegmatis or E. coli was determined. Eradication of the SD region in the absence of a downstream box abolished the translation activity. In contrast, optimized downstream box constructs resulted in a 13- and 18-fold increase in protein synthesis, relative to non-optimized DB controls in E. coli and M. smegmatis, respectively.

Base Sequence↗

PubMed2005

Expression of MADS-box genes during the embryonic phase in Arabidopsis.

MADS domain factors play important roles as developmental regulators in plants. In Arabidopsis thaliana, MADS domain proteins have been shown to regulate various processes during the vegetative and reproductive phases. Relatively little is known, however, about family members expressed during the embryonic phase and their function. To determine which MADS-box genes are expressed during the embryonic phase in Arabidopsis, a family-wide survey involving gene-specific primers and RT-PCR was conducted. Transcripts corresponding to 64 (out of 109 total) family members could be detected in RNA samples isolated from embryonic culture tissue. Eight MADS-box genes that appear to be expressed at higher levels during the embryonic phase than in seedlings or in inflorescence apices were identified. The spatial pattern of expression in developing seeds was characterized for four MADS-box genes (FLOWERING LOCUS C, FLOWERING LOCUS M, AGAMOUS-LIKE 15, and AGAMOUS-LIKE 18) using reporter constructs encoding translational fusions to GUS. All four are expressed in cells throughout the endosperm and embryo. Finally, to test the hypothesis that AGAMOUS-LIKE15 (AGL15) and AGAMOUS-LIKE18 (AGL18) play essential roles during the embryonic phase, plants carrying T-DNA insertions that disrupt these genes were isolated. No embryo defects were observed in agl15 or agl18 single mutants or in agl15agl18 double mutants. These results indicate that multiple regulatory pathways that involve MADS domain factors are likely to operate in embryonic tissues, and that genetic and/or functional redundancy are likely to be as prevalent as in other phases of the life cycle.

AGAMOUS Protein, Arabidopsis↗

PubMed1987

Localization of Xenopus homoeo-box gene transcripts during embryogenesis and in the adult nervous system.

Tissue distribution and localization of RNAs from the Xeb1 homoeo-box-containing gene were monitored with Northern blots and in situ hybridization. Xeb1 transcript distribution in larval stage embryos was established by blotting RNAs extracted from microdissected embryos. Those transcripts are restricted to a limited number of embryonic regions such as the dorsal trunk. The tissue/cell localization of Xeb1 transcripts was then monitored at several embryonic stages with in situ hybridization methods using [35S]RNA probes. These homoeo-box transcripts accumulated in a progressive and dynamic fashion. First localized in late gastrulae, they are distributed along the neural tube and in caudal mesoderm at later stages. By the swimming tadpole stage the spatial distribution of the homoeo-box transcripts is limited to specific regions of the central nervous system. Adult spinal cord shows the signal in specific neurons in the ventrolateral field of the gray matter.

Age Factors↗

PubMed1994

Calcium binding to HMG1 protein induces DNA looping by the HMG-box domains.

Electron microscopy has shown that non-histone chromosomal HMG1 could induce DNA looping or compaction in the presence (but not in the absence) of Ca2+. The effect of calcium on DNA looping and compaction was interpreted as calcium binding to the acidic C-domain of HMG1. Both individual DNA-binding HMG1-box domains A and B were found to be involved in DNA looping and compaction. Treatment of HMG1 with a thiol-specific reagent, N-ethylmaleimide, inhibited the ability of the protein to induce DNA looping and compaction but not the electrostatic interaction with DNA. These results indicated that cysteine-sulfhydryl groups of the HMG1-box domains A and B are specifically involved in DNA looping and compaction, and that in the absence of calcium the acidic C-domain down-regulates these effects by modulation of the DNA-binding properties of the HMG1-box domains.

Animals↗

PubMed1987

Transcription of Neurospora crassa 5 S rRNA genes requires a TATA box and three internal elements.

The sequences required for transcription of Neurospora crassa 5S rRNA genes have been defined using a comprehensive set of deletion and substitution mutations introduced into two cloned genes. An upstream TATA box (consensus TCATAGA) located at -29 to -24, and three internal regions (D, A and C) localized to +19 to +30, +44 to +57 and +73 to +103, respectively, are absolutely required for transcription in vitro. The TATA box fixes the start point of transcription. The A and C regions correspond to the Xenopus 5S gene internal control region but the D region has no Xenopus homologue. The spacing of the three internal elements is very strict but the position of the TATA box can be varied by up to 16 base-pairs.

Base Sequence↗