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Ovule-specific MADS-box proteins have conserved protein-protein interactions in monocot and dicot plants.

OsMADS13 is a rice MADS-box gene that is specifically expressed in developing ovules. The amino acid sequence of OsMADS13 shows 74% similarity to those of FLORAL BINDING PROTEIN 7 (FBP7) and FBP11, the products of two MADS-box genes that are necessary and sufficient to determine ovule identity in Petunia. To assess whether OsMADS13, the putative rice ortholog of FBP7 and FBP11, has an equivalent function, several analyses were performed. Ectopic expression of FBP7 and FBP11 in Petunia results in ectopic ovule formation on sepals and petals. Here we show that ectopic expression of OsMADS13 in rice and Arabidopsis does not result in the formation of such structures. Furthermore, ectopic expression of FBP7 and FBP11 in Arabidopsis also fails to induce ectopic ovule formation. To determine whether protein-protein interactions involving putative class D MADS-box proteins have been conserved, yeast two-hybrid assays were performed. These experiments resulted in the identification of three putative partners of OsMADS13, all of them encoded by AGL2-like genes. Interestingly the Petunia FBP7 protein also interacts with AGL2-like proteins. The evolutionary conservation of the MADS-box protein partners of these ovule-specific factors was confirmed by exchange experiments which showed that the protein partners of OsMADS13 interact with FBP7 and vice versa.

Germ Cells↗

Characterization and expression of 42 MADS-box genes in wheat (Triticum aestivum L.).

MADS-box genes form a large family of transcription factors and play important roles in flower development and organ differentiation in plants. In this study, 42 wheat cDNAs encoding putative MADS-box genes were isolated. BLASTX searches and phylogenetic analysis indicated that the cDNAs represented 12 of the 14 MADS-box gene subfamilies. TaAGL14 and TaAGL15 formed a new subfamily along with a rice gene OsMADS32. RT-PCR analysis revealed that these genes had different exprsssion patterns in different organs of different stages. Expression patterns of TaAGL1 and TaAGL29 were also determined using in situ hybridization. TaAGL1 was abundantly expressed in primary root tips and the whole spikelet with more intense labeling at lodicules, paleas and stamens. TaAGL29 was expressed in both the non-reproductive parts (lemma, palea and glumes), and stamens and pistils. Moreover, differential expression patterns of these genes were also observed between wheat hybrid and its parents in leaf, stem and root of jointing stage, some were up-regulated while others were down-regulated in hybrid as compared to its parents. We concluded that multiple MADS-box genes exist in wheat genome and are expressed in tissue-specific patterns, and might play important roles in wheat growth and development.

Amino Acid Sequence↗

The Hypocrea jecorina HAP 2/3/5 protein complex binds to the inverted CCAAT-box (ATTGG) within the cbh2 (cellobiohydrolase II-gene) activating element.

The 5' regulatory region of the chh2 gene, encoding cellobiohydrolase II, of the filamentous fungus Hypocrea jecorina contains the cbh2 activating element (CAE) which is essential for cbh2 expression. The CAE consists of two separate, adjacent motifs, a CCAAT box on the template strand (ATTGG) and a GTAATA box on the coding strand, which co-operate in the induction of the gene by cellulose or sophorose. EMSA supershift experiments using an antibody against Aspergillus nidulans HAPC suggested that the complex which binds to the H. jecorina CCAAT box contains a HAPC homolog. To obtain direct evidence for this, we have cloned the hap2, hap3 and hap5 genes from H. jecorina. They encode proteins whose core regions display great similarity to Aspergillus HAPB, HAPC and HAPE and to known HAP homologs from other organisms. All three genes are transcribed in a carbon source-independent manner. A. nidulans deltahap strains were functionally complemented in vitro by the overexpressed H. jecorina HAP2, HAP3 and HAP5 proteins, and they thus represent subunits of the CCAAT-binding complex. Furthermore, all three proteins (HAP2, HAP3 and HAP5) were needed to bind to the CAE in the H. jecorina cbh2 gene promoter in vitro. We conclude that the CCAAT box on the template strand in CAE is bound by the H. jecorina equivalent of the HAP protein complex.

Amino Acid Sequence↗

Laparoscopic virtual reality and box trainers: is one superior to the other?

BACKGROUND: Virtual reality (VR) simulators now have the potential to replace traditional methods of laparoscopic training. The aim of this study was to compare the VR simulator with the classical box trainer and determine whether one has advantages over the other. METHODS: Twenty four novices were tested to determine their baseline laparoscopic skills and then randomized into the following three group: LapSim, box trainer, and no training (control). After 3 weekly training sessions lasting 30-min each, all subjects were reassessed. Assessment included motion analysis and error scores. Nonparametric tests were applied, and p < 0.05 was deemed significant. RESULTS: Both trained groups made significant improvements in all parameters measured ( p < 0.05). Compared to the controls, the box trainer group performed significantly better on most of the parameters, whereas the LapSim group performed significantly better on some parameters. There were no significant differences between the LapSim and box trainer groups. CONCLUSIONS: LapSim is effective in teaching skills that are transferable to a real laparoscopic task. However, there appear to be no substantial advantages of one system over the other.

Adult↗

Fructosyltransferase mutants specify a function for the beta-fructosidase motif of the sucrose-binding box in specifying the fructan type synthesized.

The onion fructosyltransferase fructan:fructan 6G-fructosyltransferase (6G-FFT) synthesizes fructans of the inulin neo-series using 1-kestose as a substrate. 6G-FFT couples a fructosyl residue to either the terminal glucose via a beta (2-6) linkage or a terminal fructose via a beta (2-1) linkage. The sucrose-binding box is present at the N-terminus of invertases and fructosyltransferases. We tested its function by producing swaps of the first 36 amino acids of 6G-FFT with that of onion sucrose:sucrose 1-fructosyltransferase (1-SST) (SST-GFT) and vacuolar invertase (INV-GFT). In contrast to 6G-FFT, invertase and 1-SST are able to utilize sucrose as their only substrate. The chimerical enzymes were unable to use sucrose, but were active when incubated with 1-kestose. INV-GFT synthesized a similar array of fructans as 6G-FFT, in contrast, SST-GFT showed a dramatic shift in activity towards synthesis of beta (2-1) linkages. Thus the region containing the sucrose-binding box is directing the fructan type synthesized. In invertases, the beta -fructosidase motif, which is part of the sucrose-binding box, consists of NDPNG/A. This motif is variable in fructosyltransferases and consists of NDPSG in 6G-FFT and ADPNA in 1-SST of onion. We studied the importance of the 6G-FFT beta -fructosidase motif using mutants S87N (NDPNG) and N84A;S87N (ADPNG). S87N has 6G-FFT activity, whereas N84A;S87N has a activity that was shifted towards synthesis of beta (2-1) linkages. This is in agreement with the observed activities of the chimerical proteins and indicates that the beta -fructosidase motif of the sucrose-binding box is specifying the fructan type synthesized.

Amino Acid Motifs↗

The developmental expression pattern of a new murine homeo box gene: Hox-2.5.

To examine the possible role of homeo box genes in murine development we have studied the structure and expression pattern of Hox-2.5, a newly isolated homeo box gene that maps to the left end of the mouse Hox-2 locus on chromosome 11. The sequence of the Hox-2.5 homeo box has been determined. It is highly homologous to Hox-1.7 and Hox-3.2, demonstrating extended conservation among three homeo box complexes in the mouse. Northern and in situ hybridization analyses of Hox-2.5 demonstrate a novel, regionally restricted pattern of expression in developing mesoderm and neurectoderm. We detect localized Hox-2.5 transcripts as early as 8.5 days postcoitum. The expression pattern of Hox-2.5 was analyzed over the next 3 days of ontogeny, as well as in later embryonic, newborn, and adult stages. Three-dimensional reconstruction of Hox-2.5 transcript localization within the central nervous system of early embryos clearly illustrates the neural expression domain. Although the Hox-2.5 expression pattern is regionally restricted during all of these stages of development, the pattern changes along the anteroposterior and dorsoventral axes of the CNS as the embryo undergoes complex morphogenetic movements and cytodifferentiation.

Amino Acid Sequence↗

A chicken homeo box gene with developmentally regulated expression.

Several homeo box sequences were isolated from a chicken (Gallus domesticus) genomic library. One phage, lambda GG7, was chosen for further study and the single homeo box sequence it contains was characterized. The sequence of the homeo box revealed it to be a distant member of the Antp class of homeo boxes. Expression of the CHox7 gene results in four transcripts, 1.4, 1.9, 2.4 and 3.5 kb in size. Transcript accumulation of the CHox7 gene peaks twice during embryogenesis, once with the 1.4 kb transcript during early somitogenesis and the second time with the 1.9 kb transcript during organogenesis.

Amino Acid Sequence↗

A dnaA box can functionally substitute for the priming signals in the oriV of the broad host-range plasmid RSF1010.

The initiation of replication from oriV RSF1010, the replication origin of the broad host-range plasmid RSF1010, depends on RepA (helicase), RepB' (primase), and RepC (initiator protein), encoded by RSF1010 itself, while this initiation event in E. coli is independent of dnaA, dnaB, dnaC, and dnaG [Scherzinger et al. (1984) Proc. Natl. Acad. Sci. USA 81, 654-658; Scholz et al. (1985) in: Plasmids in Bacteria, pp. 243-259, Plenum, New York; Haring and Scherzinger (1989) in: Promiscuous Plasmids of Gram-negative Bacteria, pp. 95-124, Academic Press, London; Scherzinger et al. (1991) Nucl. Acids Res. 19, 1203-1211]. We showed in this work that a newly constructed origin consisting of an oriV RSF1010 and a DnaA protein binding site, the dnaA box, inserted near oriV RSF1010 (oriV RSF1010-dnaA box) could function without RepB' primase, but required RepA and RepC. This oriV RsF1010-dnaA box could not replicate in a dnaA46 strain in which only RepA and RepC were supplied, even at a permissive temperature. These results indicate that an inserted dnaA box can functionally substitute for the RSF1010-specific ssi signals, the RepB' dependent priming signals in oriV RSF1010, and can direct a priming pathway different from the RSF1010-specific one, but related to DnaA protein.

Bacterial Proteins↗

The HMG box of SRY is a calmodulin binding domain.

The HMG box domain of the testis determining factor, SRY, includes a basic amphiphilic sequence common to calmodulin (CaM) binding proteins. By affinity chromatography, native gel electrophoresis and fluorescence spectroscopy, we show the calcium-dependent binding of SRY to CaM. Binding occurs via the HMG box and an SRY peptide of residues 57-80 binds CaM like the intact domain. SRY/CaM complex formation is specifically inhibited by the SRY DNA binding site sequence, AACAAT, but not a mutated sequence. Fluorescence spectra of the SRY/CaM complex indicate 1:1 stoichiometry and that binding is accompanied by a conformational change in SRY. The A domain of HMG1 also binds CaM and we propose that CaM binding is a property of the wider HMG box family, including SOX and TCF/LEF proteins. These results suggest that CaM may regulate the DNA binding activity of HMG box transcription factors.

Amino Acid Sequence↗

The effects of estrogen on skeletal calcium metabolism and on plasma parameters of vitellogenesis in the male, three-toed box turtle (Terrapene carolina triunguis).

The box turtle (Terrapene carolina triunguis) does not appear to have evolved a mechanism for secondary bone formation similar to Aves inasmuch as several parameters of bone calcium metabolism were unaffected by estrogen treatment. This suggests that reproduction does not constitute a substantial stress to the calcium stores of the female, even though the box turtle is a terrestrial species for which a supply of calcium may be limited. However, the dermal bone of the shell of the box turtle appears to take up calcium at a rate equivalent to that of endoskeletal bones, suggesting that it participates in mineral homeostasis at least to the extent of other osseous elements. Calcium metabolism in the turtle may be unique among vertebrates in this respect. Estrogen treatment of male box turtles results in the appearance of a specific plasma protein in parallel with pronounced changes in plasma calcium and magnesium, suggesting the induction of vitellogenin. The magnitude of the vitellogenic response may have a seasonal component.

Animals↗

Reproductive behavior and LH levels of cockatiels (Nymphicus hollandicus) associated with photostimulation, nest-box presentation, and degree of mate access.

To determine the relative contribution of mate access and environmental cues in stimulating egg-laying and plasma luteinizing hormone (LH) secretion in cockatiels (Nymphicus hollandicus), and of mate access on nest-inspection and bowl-formation, mated pairs were permitted different degrees of mate access (full contact, auditory plus visual contact, auditory contact only, or no contact) and sequentially exposed to three environmental conditions (8L:16D without nest-box access for 6 weeks; 15L:9D without nest-box access for 3 weeks; 15L:9D with nest-box access for 3 weeks). The results indicate that a high degree of mate access is essential for the performance of nesting behaviors, since birds allowed no mate access or only auditory mate access were significantly less likely to inspect the nest or form a nest-bowl. Furthermore, pairs permitted no contact, auditory contact only, or auditory and visual contact delayed nest-inspection and bowl-formation and were significantly less likely to lay eggs than pairs with total mate contact. LH levels were significantly elevated only in photostimulated females of pairs of permitted full mate and nest-box access. These results suggest that maximal sexual activity in cockatiels requires an array of behavioral and environmental cues and that a linear relationship exists between extent of social cues and sexual activity.

Animals↗

A multiplicity of CCAAT box-binding proteins.

NF-Y is a sequence-specific DNA-binding protein that recognizes the Y box, a promoter element common to all major histocompatibility complex class II genes. Since the 14-base Y element harbors a CCAAT box in reverse, we were prompted to ask whether NF-Y is actually a CCAAT box-binding protein and whether it is related to the previously described CCAAT-binding factors CBP and CTF/NF-I. Data from gel retardation, methylation interference, saturation mutagenesis, and cross-competition experiments establish definitively that NF-Y is an entirely distinct CCAAT box-binding entity. Moreover, these experiments have uncovered a fourth CCAAT-binding protein, NF-Y(star) that interacts with the thymidine kinase promoter. Clearly, then, there exists a multiplicity of factors that recognize CCAAT sequences; it now becomes imperative to understand the functional significance of this multiplicity.

Animals↗

Pax 1, a member of a paired box homologous murine gene family, is expressed in segmented structures during development.

The mouse genome contains at least three copies of sequences homologous to the "paired box", a conserved domain in several Drosophila segmentation genes of the pair-rule and segment polarity classes. Overlapping phages were isolated from two different genomic libraries using the Drosophila gooseberry distal paired box as a probe. The hybridizing sequences are highly homologous to the conserved Drosophila paired box sequences. A single 3.1 kb Pax 1 (paired box gene) transcript was detected during embryonic development, whereas no transcripts were detected in adult tissues. Detailed in situ hybridization analyses with frozen embryonic sections demonstrated Pax 1 transcripts in the perichordal zone of the developing vertebral column. The expression pattern suggests a role for this gene in the formation of segmented structures of the mouse embryo.

Amino Acid Sequence↗

A long-lasting increase and decrease in synaptic excitability in the rat lateral septum are associated with high and low shuttle box performance, respectively.

In a series of experiments with rats, using evoked field potentials, the influence of massed trial training in 2-way shuttle box avoidance and step-through passive avoidance tasks was studied on the synaptic excitability of the lateral septum (LS) neurons and on the induction of long-term potentiation in the lateral septum in vivo. The majority of rats that attained a high performance level in the shuttle box task exhibited, after the shuttle box training, a long-lasting enhancement of synaptic excitability of lateral septum neurons, whereas most of the rats with low performance in the shuttle box showed a long-lasting depression in the LS synaptic excitability. Both types of excitability changes disappeared within 24 h. Neither the first habituation session in the passive avoidance apparatus nor the subsequent one-trial learning in passive avoidance task had a marked influence on lateral septum synaptic excitability. Both high-performance and low-performance rats exhibited a long-term potentiation (LTP)-like potentiation of synaptic excitability of the lateral septum neurons after high frequency stimulation of the fimbria fibers although the amount of LTP in high performance rats was slightly higher than that in low performance animals.

Animals↗

Enhancer containing unusual GC box-like sequences on the human choline acetyltransferase gene.

The minimum requirement of an enhancer for the human choline acetyltransferase (ChAT) gene was analyzed by mutagenesis and protein-DNA interaction studies. A series of deletion and site-specific mutants were expressed transiently in a rat cholinergic neuroblastoma cell, NS20Y. The results revealed that the distal region between -970 and -941 base pairs (bp) from the transcription start site is essential for efficient transcription of the human ChAT gene. Two GC box-like sequences were located within this region, and they were shown to bind purified Sp1 transcription factor by footprinting and gel retardation analyses. This sequence, however, has an orientational effect and is located approximately 1000 bp from the transcription start site, unusual for the conventional GC box sequence. Furthermore, the sequence between these GC box-like sequences is shown to bind another novel trans-acting factor by gel retardation assay and to be necessary for efficient enhancer activity. On the other hand, gel retardation assays using a nuclear extract from Drosophila SL2 cells suggested that non-Sp1 trans-acting factors could also participate in the enhancing activity of this element. Taken together, the results demonstrate that two GC box-like sequences and another trans-acting factor-binding sequence located between -970 and -941 bp act coordinately and are essential for efficient transcription of this low expressed human ChAT gene.

Animals↗

Characterisation of a functional E box motif in the proximal rat preprotachykinin-A promoter.

Three E box motifs, which are upstream of the major transcriptional start site, have previously been characterised in the rat preprotachykinin-A (rPPT) promoter. Only one of these, in the proximal promoter spanning nucleotides -67 to -47, has been demonstrated to support reporter gene expression in clonal cell lines under basal growth conditions. Here we demonstrate that the reporter gene expression can be further induced by the action of phorbol 12-myristate 13-acetate (TPA) and nerve growth factor (NGF), respectively, in both HeLa and the neuronally derived PC12 cells. This response is due to the E box motif and not an overlapping consensus sequence for a putative AP1 element, a class of element previously demonstrated to respond to both TPA and NGF in these cell lines. Finally, we demonstrate that this E box motif can support similar levels of reporter gene expression in primary cultures of dorsal root ganglion neurons as observed in clonal cell lines, demonstrating that E box binding complexes can (1) function as a transcriptional regulator in dorsal root ganglion neurons and (2) bind to and therefore presumably regulate rPPT promoter activity.

Animals↗

A H1 histone gene-specific AC-box-related element influences transcription from a major chicken H1 promoter.

In comparing several histone H1 promoters, we have identified a highly conserved sequence element, 5'-TGTGTTA, located approx. 450-480 bp upstream from the cap site. This TG-box is a near perfect inverted repeat of the previously characterized AC-box (5'-AAACACA). The distance between these elements is also highly conserved. We performed transient transfection assays with cat gene reporter constructs which indicated that both the presence and correct position of the TG-box were essential for maximal expression of the chicken 02 H1 promoter. To the best of our knowledge, this study represents the first demonstration of an effect by the TG-box on transcription of a major histone-encoding H1 gene.

Animals↗

Pax: a murine multigene family of paired box-containing genes.

A murine multigene family has been identified that shares a conserved sequence motif, the paired box, with developmental control and tissue-specific genes of Drosophila. To date five murine paired box-containing genes (Pax genes) have been described and one, Pax-1, has been associated with the developmental mutant phenotype undulated. Here we describe the paired boxes of three novel Pax genes, Pax-4, Pax-5, and Pax-6. Comparison of the eight murine paired domains of the mouse, the five Drosophila paired domains, and the three human paired domains shows that they fall into six distinct classes: class I comprises Pox meso, Pax-1, and HuP48; class II paired, gooseberry-proximal, gooseberry-distal, Pax-3, Pax-7, HuP1, and HuP2; class III Pax-2, Pax-5, and Pax-8; class IV Pax-4; class V Pox neuro; and class VI Pax-6. Pax-1 and the human gene HuP48 have identical paired domains, as do Pax-3 and HuP2 as well as Pax-7 and HuP1, and are likely to represent homologous genes in mouse and man. Identical intron-exon structure and extensive sequence homology of their paired boxes suggest that several Pax genes represent paralogs. The chromosomal location of all novel Pax genes and of Pax-3 and Pax-7 has been determined and reveals that they are not clustered.

Amino Acid Sequence↗