The cytokinin receptors of Arabidopsis are located mainly to the endoplasmic reticulum

The plant hormone cytokinin is perceived by membrane-located sensor histidine kinases. Arabidopsis (Arabidopsis thaliana) possesses three cytokinin receptors: ARABIDOPSIS HISTIDINE KINASE2 (AHK2), AHK3, and CYTOKININ RESPONSE1/AHK4. The current model predicts perception of the cytokinin signal at the plasma membrane. However, cytokinin-binding studies with membrane fractions separated by two-phase partitioning showed that in the wild type, as well as in mutants retaining only single cytokinin receptors, the major part of specific cytokinin binding was associated with endomembranes. Leaf epidermal cells of tobacco (Nicotiana benthamiana) expressing receptor-green fluorescent protein fusion proteins and bimolecular fluorescence complementation analysis showed strong fluorescence of the endoplasmic reticulum (ER) network for all three receptors. Furthermore, separation of the microsomal fraction of Arabidopsis plants expressing Myc-tagged AHK2 and AHK3 receptors by sucrose gradient centrifugation followed by immunoblotting displayed the Mg2?-dependent density shift typical of ER membrane proteins. Cytokinin-binding assays, fluorescent fusion proteins, and biochemical fractionation all showed that the large majority of cytokinin receptors are localized to the ER, suggesting a central role of this compartment in cytokinin signaling. A modified model for cytokinin signaling is proposed.     

The most frequent short sequences in non-coding DNA

The purpose of this work is to determine the most frequent short sequences in non-coding DNA. They may play a role in maintaining the structure and function of eukaryotic chromosomes. We present a simple method for the detection and analysis of such sequences in several genomes, including Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens. We also study two chromosomes of man and mouse with a length similar to the whole genomes of the other species. We provide a list of the most common sequences of 9–14 bases in each genome. As expected, they are present in human Alu sequences. Our programs may also give a graph and a list of their position in the genome. Detection of clusters is also possible. In most cases, these sequences contain few alternating regions. Their intrinsic structure and their influence on nucleosome formation are not known. In particular, we have found new features of short sequences in C. elegans, which are distributed in heterogeneous clusters. They appear as punctuation marks in the chromosomes. Such clusters are not found in either A. thaliana or D. melanogaster. We discuss the possibility that they play a role in centromere function and homolog recognition in meiosis.     

The distribution of RNA motifs in natural sequences.

Functional analysis of genome sequences has largely ignored RNA genes and their structures. We introduce here the notion of 'ribonomics' to describe the search for the distribution of and eventually the determination of the physiological roles of these RNA structures found in the sequence databases. The utility of this approach is illustrated here by the identification in the GenBank database of RNA motifs having known binding or chemical activity. The frequency of these motifs indicates that most have originated from evolutionary drift and are selectively neutral. On the other hand, their distribution among species and their location within genes suggest that the destiny of these motifs may be more elaborate. For example, the hammerhead motif has a skewed organismal presence, is phylogenetically stable and recent work on a schistosome version confirms its in vivo biological activity. The under-representation of the valine-binding motif and the Rev-binding element in GenBank hints at a detrimental effect on cell growth or viability. Data on the presence and the location of these motifs may provide critical guidance in the design of experiments directed towards the understanding and the manipulation of RNA complexes and activities in vivo.     

The role of peptide motifs in the evolution of a protein network

Naturally occurring proteins in cellular networks often share peptide motifs. These motifs have been known to play a pivotal role in protein interactions among the components of a network. However, it remains unknown how these motifs have contributed to the evolution of the protein network. Here we addressed this issue by a synthetic biology approach. Through the motif programming method, we have constructed an artificial protein library by mixing four peptide motifs shared among the Bcl-2 family proteins that positively or negatively regulate the apoptosis networks. We found one strong pro-apoptotic protein, d29, and two proteins having moderate, but unambiguous anti-apoptotic functions, a10 and d16, from the 28 tested clones. Thus both the pro- and anti-apoptotic modulators were present in the library, demonstrating that functional proteins with opposing effects can emerge from a single pool prepared from common motifs. Motif programming studies have exhibited that the annotated function of the motifs were significantly influenced by the context that the motifs embedded. The results further revealed that reshuffling of a set of motifs realized the promiscuous state of protein, from which disparate functions could emerge. Our finding suggests that motifs contributed to the plastic evolvability of the protein network.     

Light regulatory sequences are located within the 5' portion of the Fed-1 message sequence.

We have previously shown that element(s) mediating a light-induced increase in the abundance of Fed-1 mRNA in the leaves of transgenic tobacco plants are located within the transcribed portion of the gene. As part of an effort to define the mechanism of this effect, we report here that cis-acting elements capable of mediating a 5-fold light-induced increase in the abundance of this mRNA are located within a region comprising the 5' leader and first third of the Fed-1 coding sequence. No activity was detected in the 3' untranslated region of the gene. In a gain-of-function assay, the 5' region was found to be capable of conferring light responsiveness on three different reporter sequences, although experiments with the gusA reporter were complicated by an apparent negative light effect on the stability of this mRNA. Deletion experiments show that at least one essential light regulatory element is located in the 5' untranslated region of Fed-1 between nucleotides +19 and +57. Additional Fed-1 sequences, including a portion of the protein coding region, are required to confer positive responsiveness on the gusA reporter. These additional sequences may include specific light regulatory elements or simply provide an environment in which the leader element can function normally.     

Bovine thymus satellite I DNA sequences, which are highly methylated, are preferentially located in H1-rich chromatin

The distribution of 5-methylcytosine among H1-rich and -poor bovine thymus chromatin regions was determined. 5-Methylcytosine was enriched in H1-rich chromatin regions, with linker and nucleosomal DNA containing similar amounts of this modified base. Satellite I DNA sequences, which constitute 5-7% of the genome and are highly methylated, were preferentially localized among H1-rich chromatin regions, in accordance with the distribution of 5-methylcytosine. In contrast to the satellite I DNA sequences, prothrombin (a single copy DNA sequence) was localized among both H1-rich and -poor chromatin regions. The results of this study are consistent with the hypothesis that DNA methylation has a role in modulating the structure of chromatin.     

Certain heptapeptide and large sequences representing an entire helix, strand or coil conformation in proteins are associated as chameleon sequences

Helices, strands and coils in proteins of known three-dimensional structure, corresponding to heptapeptide and large sequences (‘probe’ peptides), were scanned against peptide sequences of variable length, comprising seven or more residues that correspond to a different conformation (‘target’ peptides) in protein crystal structures available from the Protein Data Bank (PDB). Where the ‘probe’ and ‘target’ peptide sequences exactly match, they correspond to ‘chameleon’ sequences in protein structures. We observed ∼548 heptapeptide and large chameleon sequences that included peptides in the coil conformation from 53,794 PDB files that were analyzed. However, after excluding several chameleon peptides based on the quality of protein structure data, redundancy and peptides associated with cloning artifacts, such as, histidine-tags, we observed only ten chameleon peptides in structurally different proteins and the maximum length comprised seven amino acid residues. Our analysis suggests that the quality of protein structure data is important for identifying possibly, the ‘true chameleons’ in PDB. Majority of the chameleon sequences correspond to an entire strand in one protein that is observed as part of helix sequence in another protein. The heptapeptide chameleons are characterized with a high propensity of alanine, leucine and valine amino acid residues. The total hydropathy values range between −11.2 and 22.9, the difference in solvent accessibility between 2.0 Å² and 373 Å² units and the difference in total number of residue neighbor contacts between 0 and 7 residues. Our work identifies for the first time heptapeptide and large sequences that correspond to a single complete helix, strand or coil, which adopt entirely different secondary structures in another protein.     

Non-neutral processes drive the nucleotide composition of non-coding sequences in Drosophila

The nature of the forces affecting base composition is a key question in genome evolution. There is uncertainty as to whether differences in the GC contents of non-coding sequences reflect differences in mutational bias, or in the intensity of selection or biased gene conversion. We have used a polymorphism dataset for non-coding sequences on the X chromosome of Drosophila simulans to examine this question. The proportion of GC-->AT versus AT-->GC polymorphic mutations in a locus is correlated with its GC content. This implies the action of forces that favour GC over AT base pairs, which are apparently strongest in GC-rich sequences.     

Long non-coding RNAs in wheat are related to its susceptibility to powdery mildew

To identify genes involved in the susceptibility to powdery mildew in wheat, genechip hybridization experiments were performed applying interactions between resistant and susceptible wheat cultivars and powdery mildew. Ten genes were found to express exclusively in the susceptible host after inoculation. The single cell transient expression experiments revealed that the RNA interference (RNAi) of two expressed sequence tag (EST) sequences (accession No. BQ168479 and CA648596) could enhance host resistance by 22 and 15 %, respectively. Full-length cDNA cloning revealed that they represented long non-coding RNAs (lncRNAs) and markedly accumulated during the powdery mildew primary infection.     

Folding of a peptide corresponding to the alpha-helix in bovine pancreatic trypsin inhibitor

A short peptide corresponding to the alpha-helical region of BPTI shows partial folding in aqueous solution (pH 7) as judged by circular dichroism (CD). Folding is temperature and denaturant sensitive, and the peptide is monomeric. The difference CD spectrum, obtained from spectra at two temperatures, indicates that the peptide folds as an alpha-helix. Difference CD spectroscopy provides a sensitive assay for helix formation in peptides exhibiting small amounts of structure. Helix stability in this peptide shows a marked pH dependence which is consistent with stabilizing charged side-chain interactions with the helix dipole and/or salt bridge formation.     

The mammalian homologues of frog Bv8 are mainly expressed in spermatocytes

Bv8, a protein from skin secretions of Bombina variegata, reacts with receptors present in mammalian brain and intestine (Mollay et al. (1999) Eur. J. Pharmacol. 374, 189-196). As deduced from cloned cDNAs, the murine and human Bv8 homologues have identical amino-terminal sequences and also contain 10 cysteines. From mouse testes, two forms of Bv8 mRNA have been characterized, of which one contains an additional exon which codes for 21 mostly basic amino acids. The mouse Bv8 gene is most active in mid-late pachytene spermatocytes. In mouse testes, Bv8 mRNA can first be detected at the end of the second week post partum.     

A model-based approach to study nearest-neighbor influences reveals complex substitution patterns in non-coding sequences

In this article, we present a likelihood-based framework for modeling site dependencies. Our approach builds upon standard evolutionary models but incorporates site dependencies across the entire tree by letting the evolutionary parameters in these models depend upon the ancestral states at the neighboring sites. It thus avoids the need for introducing new and high-dimensional evolutionary models for site-dependent evolution. We propose a Markov chain Monte Carlo approach with data augmentation to infer the evolutionary parameters under our model. Although our approach allows for wide-ranging site dependencies, we illustrate its use, in two non-coding datasets, in the case of nearest-neighbor dependencies (i.e., evolution directly depending only upon the immediate flanking sites). The results reveal that the general time-reversible model with nearest-neighbor dependencies substantially improves the fit to the data as compared to the corresponding model with site independence. Using the parameter estimates from our model, we elaborate on the importance of the 5-methylcytosine deamination process (i.e., the CpG effect) and show that this process also depends upon the 5' neighboring base identity. We hint at the possibility of a so-called TpA effect and show that the observed substitution behavior is very complex in the light of dinucleotide estimates. We also discuss the presence of CpG effects in a nuclear small subunit dataset and find significant evidence that evolutionary models incorporating context-dependent effects perform substantially better than independent-site models and in some cases even outperform models that incorporate varying rates across sites.