Odorant receptors of a primitive hymenopteran pest, the wheat stem sawfly.

The wheat stem sawfly, Cephus cinctus, is an herbivorous hymenopteran that feeds exclusively on members of the Graminae family. Synanthropically, it has become one of the most important insect pests of wheat grown in the northern Great Plains region of the USA and Canada. Insecticides are generally ineffective because of the wheat stem sawfly's extended adult flight period and its inaccessible larval stage, during which it feeds within the wheat stems, making it virtually intractable to most pest management strategies. While research towards integrated pest management strategies based on insect olfaction has proved promising, nothing is known about the molecular basis of olfaction in this important pest species. In this study we identified 28 unique odorant receptor (Or) transcripts from an antennal transcriptome. A phylogenetic analysis with the predicted Ors from the honey bee and jewel wasp genomes revealed at least four clades conserved amongst all three Hymenoptera species. Antennal expression levels were analysed using quantitative real-time PCR, and one male-biased and five female-biased Ors were identified. This study provides the basis for future functional analyses to identify behaviourally active odours that can be used to help develop olfactory-mediated pest management tools.

Terminal oxidase diversity and function in "Metallosphaera yellowstonensis": gene expression and protein modeling suggest mechanisms of Fe(II) oxidation in the sulfolobales.

"Metallosphaera yellowstonensis" is a thermoacidophilic archaeon isolated from Yellowstone National Park that is capable of autotrophic growth using Fe(II), elemental S, or pyrite as electron donors. Analysis of the draft genome sequence from M. yellowstonensis strain MK1 revealed seven different copies of heme copper oxidases (subunit I) in a total of five different terminal oxidase complexes, including doxBCEF, foxABCDEFGHIJ, soxABC, and the soxM supercomplex, as well as a novel hypothetical two-protein doxB-like polyferredoxin complex. Other genes found in M. yellowstonensis with possible roles in S and or Fe cycling include a thiosulfate oxidase (tqoAB), a sulfite oxidase (som), a cbsA cytochrome b(558/566), several small blue copper proteins, and a novel gene sequence coding for a putative multicopper oxidase (Mco). Results from gene expression studies, including reverse transcriptase (RT) quantitative PCR (qPCR) of cultures grown autotrophically on either Fe(II), pyrite, or elemental S showed that the fox gene cluster and mco are highly expressed under conditions where Fe(II) is an electron donor. Metagenome sequence and gene expression studies of Fe-oxide mats confirmed the importance of fox genes (e.g., foxA and foxC) and mco under Fe(II)-oxidizing conditions. Protein modeling of FoxC suggests a novel lysine-lysine or lysine-arginine heme B binding domain, indicating that it is likely the cytochrome component of a heterodimer complex with foxG as a ferredoxin subunit. Analysis of mco shows that it encodes a novel multicopper blue protein with two plastocyanin type I copper domains that may play a role in the transfer of electrons within the Fox protein complex. An understanding of metabolic pathways involved in aerobic iron and sulfur oxidation in Sulfolobales has broad implications for understanding the evolution and niche diversification of these thermophiles as well as practical applications in fields such as bioleaching of trace metals from pyritic ores.

Chromatin silencing protein and pachytene checkpoint regulator Dot1p has a methyltransferase fold.

Although protein methylation has been observed for decades, its functional significance has remained largely unclear. Using sensitive profile searches and structural modeling, chromatin silencing protein and meiotic recombination checkpoint regulator Dot1p was identified as a putative protein methyltransferase. Along with recent results that link histone H3 methylation with chromatin silencing, this finding suggests that an expanded combinatorial repertoire of protein modifications affects transcriptional regulation.

DNA sequence-dependent folding determines the divergence in binding specificities between Maf and other bZIP proteins.

Maf family transcription factors are atypical basic region-leucine zipper (bZIP) proteins that contain a variant basic region and an ancillary DNA-binding region. These proteins recognize extended DNA sequence elements flanking the core recognition element bound by canonical bZIP proteins. We have investigated the causes for the differences in DNA recognition between Maf and other bZIP family proteins through studies of Maf secondary structure, trypsin sensitivity, binding affinity, dissociation rate and DNA contacts. Our results show that specific DNA binding by Maf is coupled to a conformational change involving both the basic and ancillary DNA-binding regions that depends on the extended DNA sequence elements. Two basic region amino acid residues that differ between Maf and canonical bZIP proteins facilitate the conformational change required for Maf recognition of the extended elements. Nucleotide base contacts made by Maf differ from those made by canonical bZIP proteins. Taken together, our results suggest that the unusual DNA binding specificity of Maf family proteins is mediated by concerted folding of structurally unrelated DNA recognition motifs.

Identification and cDNA cloning of a novel RNA-binding protein that interacts with the cyclic nucleotide-responsive sequence in the Type-1 plasminogen activator inhibitor mRNA.

Incubation of HTC rat hepatoma cells with 8-bromo-cAMP results in a 3-fold increase in the rate of degradation of type-1 plasminogen activator inhibitor (PAI-1) mRNA. We have reported previously that the 3'-most 134 nt of the PAI-1 mRNA is able to confer cyclic nucleotide regulation of message stability onto a heterologous transcript. R-EMSA and UV cross-linking experiments have shown that this 134 nt cyclic nucleotide-responsive sequence (CRS) binds HTC cell cytoplasmic proteins ranging in size from 38 to 76 kDa. Mutations in the A-rich region of the CRS both eliminate cyclic nucleotide regulation of mRNA decay and abolish RN-protein complex formation, suggesting that these RNA-binding proteins may be important regulators of mRNA stability. By sequential R-EMSA and SDS-PAGE we have purified a protein from HTC cell polysomes that binds to the PAI-1 CRS. N-terminal sequence analysis and a search of protein data bases revealed identity with two human sequences of unknown function. We have expressed one of these sequences in E. coli and confirmed that the recombinant protein interacts specifically with the PAI-1 CRS. Mutation of the A-rich portion of the PAI-1 CRS reduces binding by the recombinant PAI-1 RNA-binding protein. The amino acid sequence of this protein includes an RGG box and two arginine-rich regions, but does not include other recognizable RNA binding motifs. Detailed analyses of nucleic acid and protein data bases demonstrate that blocks of this sequence are highly conserved in a number of metazoans, including Arabidopsis, Drosophila, birds, and mammals. Thus, we have described a novel RNA-binding protein that identifies a family of proteins with a previously undefined sequence motif. Our results suggest that this protein, PAI-RBP1, may play a role in regulation of mRNA stability.

Unconventional helical phasing of repetitive DNA motifs reveals their relative bending contributions.

A novel, multiple DNA phasing analysis is described in which three sequence motifs associated with bent DNA are clustered together in oligomers of identical base composition, but with different phasing relationships of these motifs to each other. Synthetic oligonucleotides containing different combinations of AAAAA(A), GGGCCC and GAGAG sequence motifs were ligated and analyzed by gel mobility and cyclization experiments to determine their global curvature. These assays were used to obtain relative bending contributions of the analyzed sequence motifs. The experimental results also provide a rigorous test of predictive models for DNA bending. We report, using molecular modeling, that none of the most widely used dinucleotide (nearest neighbor) models can accurately describe the conformational properties of these DNA sequences and that more complex models, at least at the trinucleotide level, are required.

DIAMOD: display and modeling of DNA bending.

MOTIVATION: DIAMOD (Displayandmodeling ofDNA) was created as a user-friendly software for exploring and better understanding DNA structural variations, particularly DNA bending. It was intended to be as open as possible so that any of the existing or future predictive models can be used with it. RESULTS: DIAMOD features graphic display and interactive manipulation of DNA molecules on the screen. Since it works with di-, tri- or tetranucleotide models supplied as external files of angular parameters, it was recently used to evaluate critically all available predictive models for DNA bending. The program has a unique option to insert bends at defined positions in DNA sequence independently of the currently used model, which enables the simulation of both intrinsic and protein-induced kinking. Finally, many output file formats facilitate the sharing of data with other programs and the creation of visually pleasing images. AVAILABILITY: The program is available on request to academic users free of charge. It will be distributed via the WWW (http://www-personal.umich.edu/ mensur/software.html). Users with no network access can get a copy directly from the author. CONTACT: mensur@umich.edu

Strained DNA is kinked by low concentrations of Zn2+.

A novel atomic force microscope with a magnetically oscillated tip has provided unprecedented resolution of small DNA fragments spontaneously adsorbed to mica and imaged in situ in the presence of divalent ions. Kinks (localized bends of average angle 78 degrees) were observed in axially strained minicircles consisting of tandemly repeated d(A)5 and d(GGGCC[C]) sequences. The frequency of kinks in identical minicircles increased 4-fold in the presence of 1 mM Zn2+ compared with 1 mM Mg2+. Kinking persisted in mixed Mg2+/Zn2+ electrolytes until the Zn2+ concentration dropped below 100 microM, indicating that this type of kinking may occur under physiological conditions. Kinking appears to replace intrinsic bending, and statistical analysis shows that kinks are not localized within any single sequence element. A surprisingly small free energy is associated with kink formation.

The organic crystallizing agent 2-methyl-2,4-pentanediol reduces DNA curvature by means of structural changes in A-tracts.

Contemporary predictive models for sequence-dependent DNA structure provide a good estimation of overall DNA curvature in most cases. However, the two current models differ fundamentally in their view of the origin of DNA curvature. An earlier model that associates DNA bending primarily, although not exclusively, with stretches of adenines (A-tracts) is based on results of comparative gel retardation, cyclization kinetics, hydroxyl radical cutting, and other solution measurements. It represents an intersection of wedge and junction models. More recently, a non-A-tract bending model has been proposed, built on structural results from x-ray crystallography and molecular modeling. In this view, A-tracts are proposed to be straight and rigid, whereas mixed sequence DNA is bent. Because a key premise of the non-A-tract bending model is the crystallographic observation that A-tracts are straight, we have examined the effect in solution of 2-methyl-2,4-pentanediol (MPD), an organic solvent used in crystal preparation for crystallographic DNA structure determinations. Using cyclization analysis, DNase I cutting, chemical probing, and electron microscopy on DNA oligomers with and without A-tracts, we show that the presence of MPD in solution dramatically affects A-tracts and that the effect is specific to these sequence elements. Combined with the previous observation that MPD affects gel mobility of curved sequences with A-tracts, our findings support the bent A-tract model and call for caution in the interpretation of crystallographic results on DNA structure as these are presently obtained.

The effects of sequence context on DNA curvature.

Recent experiments have exposed significant discrepancies between experimental data and predictive models for DNA structure. These results strongly suggest that DNA structural parameters incorporated in the models are not always sufficient to account for the influence of sequence context and of specific ion effects. In an attempt to evaluate these two effects, we have investigated repetitive DNA sequences with the sequence motif GAGAG.CTCTC located in different helical phasing arrangements with respect to poly(A) tracts and GGGCCC.GGGCCC sequence motifs. Methods used are ligase-mediated cyclization and gel mobility experiments along with DNase I cutting and chemical probe studies. The results provide new evidence for curvature in poly(A) tracts. They also show that the sequence context in which bending and flexible sequence elements are found is an important aspect of sequence-dependent DNA conformation. Although dinucleotide models generally have good predictive power, this work demonstrates that in some instances sequence elements larger than the dinucleotide must be taken into account, and hence it provides a starting point for the appropriate modification and refinement of existing structural models for DNA.

Bending and torsional flexibility of G/C-rich sequences as determined by cyclization assays.

The structural polymorphism of DNA is a vital aspect of its biological function. However, it has become increasingly apparent in recent years that DNA polymorphism is a complicated, multidimensional phenomenon that includes not only static sequence-directed structures but dynamic effects as well, including influences of counterions and sequence context. In order to address some of these additional factors that govern DNA conformation, we have used T4 ligase-mediated cyclization to investigate bending in a series of DNA sequences containing the GGGCCC.GGGCCC motif in different sequence contexts including various helical phasings with (A)5-tracts. We present evidence for curvature in GGGCCC.GGGCCC and (A)5-tract motifs in the presence of physiological levels of Mg2+ and show that these motifs curve through similar but oppositely directed bending angles under these ionic strength conditions. Although these two sequence motifs appear to bend similarly, our results suggest significant differences in stiffness and stability of curvature between them. We also show that under the same experimental conditions, the CTAG-CTAG sequence element possesses unusual torsional flexibility and that this appears to be associated with the central TA.TA dinucleotide. The results underscore the need to include sequence context and specific ion effects as well as a dynamic basis in more complete predictive models for functionally related DNA polymorphism.

What is the basis of sequence-directed curvature in DNAs containing A tracts?

A variety of solution and gel experiments show that DNAs containing tracts of 4-8 A's repeated in phase with the helix repeat are curved. Several independent analyses of these experiments argue that curvature resides in the A tracts themselves. In x-ray crystallographic studies of several DNAs containing A tracts, however, the A tracts are uncurved, leading to models in which curvature resides in the non-A tracts. This "curved general sequence model" has several problems, in our view. We review those, and we describe recent experiments that show that the dehydrating agents commonly used in x-ray crystallography markedly reduce curvature in gels and in solution, calling into question the ability of crystallography to determine the structural basis of DNA curvature. Finally, we discuss the critical role of hydration in curved DNAs and suggest new experiments that we hope could finally determine exactly which sequences are responsible for curvature.

Physiological concentration of magnesium ions induces a strong macroscopic curvature in GGGCCC-containing DNA.

The bending propensity of non-A/T DNA sequence elements is well known, but helical phasing/gel mobility experiments fail to reveal an intensive macroscopic curvature if A/T tracts are not present in the sequence. Recent X-ray data prove on the other hand that a GGCC element is intrinsically curved toward the major groove, which seemingly contradicts the fact that macroscopic curvature at GGGCCC elements is hardly detectable with a conventional gel mobility assay. Here we show that GGGCCC containing DNA, with no A/T tracts in the sequence context, has a detectable, strong gel mobility anomaly only in the presence of divalent ions (10 mM Mg2+ or Ca2+, 1 mM Zn2+). Metal ions increase the gel mobility anomaly in A/T tracts as well, but the effect is substantially stronger for GGGCCC than for the rigid A/T tracts. Our data suggest that metal ions change the sequence-dependent dynamic features of DNA; on the other hand, there is no evidence of twist-mediated change of the planarity of curvature in the presence of metal ions. The results show that near-physiological concentrations of divalent cations (10 mM MgCl2) have a strong and differential effect on various sequence elements, so that the current picture of sequence-dependent DNA curvature is changed not only in a quantitative, but also in a qualitative sense.

Evidence for opposite groove-directed curvature of GGGCCC and AAAAA sequence elements.

The repetitive sequence (AGGGCCCTAGAGGGGCCC-TAG)n was previously shown to be curved by gel mobility assays. Here we show, using hydroxy radical/DNase I digestion and differential helical phasing experiments that the curvature is directed towards the major groove and is located in the GGGCCC, but not the CTAGAG segments. The effect of the GC step in the context of the GGGCCC motif is apparently about as large as that of AA/TT, i.e. enough to cancel the macroscopic curvature of helically phased A-tracts. These data are in agreement with positive roll-like curvature of the GCC/GGC motif, predicted from nucleosome packing data and the 3D structure of the GGGGCCCC octamer, but they are not in agreement with the dinucleotide-based roll angle values predicted for AG/CT, TA, GG/CC and GC steps. Our results thus indicate the importance of interactions beyond the dinucleotide steps in predictive models of DNA curvature.