Unusual pheromone chemistry in the navel orangeworm: novel sex attractants and a behavioral antagonist.

Using molecular- and sensory physiology-based approaches, three novel natural products, a simple ester, and a behavioral antagonist have been identified from the pheromone gland of the navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae). In addition to the previously identified (Z,Z)-11,13-hexadecadienal, the pheromone blend is composed of (Z,Z,Z,Z,Z)-3,6,9,12,15-tricosapentaene, (Z,Z,Z,Z,Z)-3,6,9,12,15-pentacosapentaene, ethyl palmitate, ethyl-(Z,Z)-11,13-hexadecadienoate, and (Z,Z)-11,13-hexadecadien-1-yl acetate. The C(23) and C(25) pentaenes are not only novel sex pheromones, but also new natural products. In field tests, catches of A. transitella males in traps baited with the full mixture of pheromones were as high as those in traps with virgin females, whereas control and traps baited only with the previously known constituent did not capture any moths at all. The navel orangeworm sex pheromone is also an attractant for the meal moth, Pyralis farinalis L. (Pyralidae), but (Z,Z)-11,13-hexadecadien-1-yl acetate is a behavioral antagonist. The new pheromone blend may be highly effective in mating disruption and monitoring programs.

Taste receptor cells express pH-sensitive leak K+ channels.

Two-pore domain K+ channels encoded by genes KCNK1-17 (K2p1-17) play important roles in regulating cell excitability. We report here that rat taste receptor cells (TRCs) highly express TASK-2 (KCNK5; K2p5.1), and to a much lesser extent TALK-1 (KCNK16; K2p16.1) and TASK-1 (KCNK3; K2p3.1), and suggest potentially important roles for these channels in setting resting membrane potentials and in sour taste transduction. Whole cell recordings of isolated TRCs show that a leak K+ (Kleak) current in a subset of TRCs exhibited high sensitivity to acidic extracellular pH similar to reported properties of TASK-2 and TALK-1 channels. A drop in bath pH from 7.4 to 6 suppressed 90% of the current, resulting in membrane depolarization. K+ channel blockers, BaCl2, but not tetraethylammonium (TEA), inhibited the current. Interestingly, resting potentials of these TRCs averaged -70 mV, which closely correlated with the amplitude of the pH-sensitive Kleak, suggesting a dominant role of this conductance in setting resting potentials. RT-PCR assays followed by sequencing of PCR products showed that TASK-1, TASK-2, and a functionally similar channel, TALK-1, were expressed in all three types of lingual taste buds. To verify expression of TASK channels, we labeled taste tissue with antibodies against TASK-1, TASK-2, and TASK-3. Strong labeling was seen in some TRCs with antibody against TASK-2 but not TASK-1 and TASK-3. Consistent with the immunocytochemical staining, quantitative real-time PCR assays showed that the message for TASK-2 was expressed at significantly higher levels (10-100 times greater) than was TASK-1, TALK-1, or TASK-3. Thus several K2P channels, and in particular TASK-2, are expressed in rat TRCs, where they may contribute to the establishment of resting potentials and sour reception.

Characterization of the transvection mediating region of the abdominal-B locus in Drosophila.

Genetic studies have identified an unusual transvection process in the Abdominal-B (Abd-B) locus of Drosophila. In some cases distal infraabdominal (iab) regulatory domains continue to activate the Abd-B promoter even when translocated onto different chromosomes. Transvection depends on an approx. 10 kb genomic DNA sequence, termed the transvection mediating region (tmr), located immediately downstream of the Abd-B transcription unit. Here we report a detailed analysis of this region. Different DNA fragments from the tmr were inserted into a variety of P-transformation vectors. Analyses of reporter gene expression in transgenic embryos and adults identify at least three cis-regulatory elements, including two enhancers (IAB7 and IAB8) and a new insulator DNA (Frontabdominal-8, Fab-8). Evidence is also presented for a Polycomb Response Element (PRE) linked to the IAB8 enhancer, and an internal promoter in the iab-8 domain, which transcribes the iab-7 and iab-8 cis-regulatory DNA, including the Fab-8 insulator. We discuss the significance of these findings with regard to Abd-B transvection and long-range enhancer-promoter interactions in mammalian globin loci.

Molecular Biology Database List.

Molecular Biology Database List (MBDL) includes brief descriptions and pointers to Web sites for the various databases described in this issue as well as other Web sites presenting data sets relevant to molecular biology. This information is compiled into a list (http://www.oup.co.uk/nar/Volume_27/Issue_01/summary/ gkc105_gml.html) which includes links both to source Web sites and to on-line versions of articles describing the databases.

The K box, a conserved 3' UTR sequence motif, negatively regulates accumulation of enhancer of split complex transcripts.

Cell-cell interactions mediated by the Notch receptor play an essential role in the development of the Drosophila adult peripheral nervous system (PNS). Transcriptional activation of multiple genes of the Enhancer of split Complex [E(spl)-C] is a key intracellular response to Notch receptor activity. Here we report that most E(spl)-C genes contain a novel sequence motif, the K box (TGTGAT), in their 3' untranslated regions (3' UTRs). We present three lines of evidence that demonstrate the importance of this element in the post-transcriptional regulation of E(spl)-C genes. First, K box sequences are specifically conserved in the orthologs of two structurally distinct E(spl)-C genes (m4 and m8) from a distantly related Drosophila species. Second, the wild-type m8 3' UTR strongly reduces accumulation of heterologous transcripts in vivo, an activity that requires its K box sequences. Finally, m8 genomic DNA transgenes lacking these motifs cause mild gain-of-function PNS defects and can partially phenocopy the genetic interaction of E(spl)D with Notchspl. Although E(spl)-C genes are expressed in temporally and spatially specific patterns, we find that K box-mediated regulation is ubiquitous, implying that other targets of this activity may exist. In support of this, we present sequence analyses that implicate genes of the iroquois Complex (Iro-C) and engrailed as additional targets of K box-mediated regulation.

Partial purification of plasma phenoloxidase of the yellow fever mosquito Aedes aegypti L. (Diptera: Culicidae).

A nitrocellulose-based assay was developed using a dot-blot apparatus to detect phenoloxidase activity in column fractions. Using this assay, plasma phenoloxidase was partially purified from Aedes aegypti larvae using hydrophobic interaction chromatography, gel filtration, and ion-exchange chromatography. The molecular weight (M(r)) native enzyme was 130,000, and it contained subunits of 76,000, 62,000, and 58,000. Two phenoloxidase peaks were observed by ion exchange chromatography, and these fractions had distinct polypeptide profiles as detected by SDS-PAGE.

DNA structural patterns and nucleosome positioning.

There is no clear picture to date of the mechanisms determining nucleosome positioning. Generally, local DNA sequence signals (sequence-dependent positioning) or non-local signals (e.g. boundary effects) are possible. We have analyzed the DNA sequences of a series of positioned and mapped nucleosome cores in a systematic search for local sequence signals. The data set consists of 113 mapped nucleosome cores, mapped in vivo, in situ, or in reconstituted chromatin. The analysis focuses on the periodic distribution of sequence elements implied by each of six different published DNA structural models. We have also investigated the periodic distribution of all mono-, di-, and trinucleotides. An identical analysis was performed on a set of isolated chicken nucleosome cores (nucleosome data from the literature) that are presumably positioned due to local sequence signals. The results show that the sequences of the isolated nucleosome cores have a number of characteristic features that distinguish them clearly from randomly chosen reference DNA. This confirms that the positioning of these nucleosomes is mainly sequence-dependent (i.e., dependent on local octamer-DNA interactions) and that our algorithms are able to detect these patterns. Using the same algorithms, the sequences of the mapped nucleosome cores, however, are on average very similar to randomly chosen reference DNA. This suggests that the position of the majority of these nucleosomes can not be attributed to the sequence patterns implemented in our algorithms. The arrangement of positioned nucleosomes seems to be the result of a dynamic interplay of octamer-DNA interactions, nucleosome-nucleosome interactions and other positioning signals with varying relative contributions along the DNA.

GRAM and genfragII: solving and testing the single-digest, partially ordered restriction map problem.

GRAM (Genomic Restriction map AsseMbly) takes as input single-digest restriction fragments for a set of overlapping clones and outputs one or more plausible partially ordered restriction maps. For each restriction map, GRAM shows the corresponding alignment of the input clone fragments. Due to the error and uncertainty in experimental data, this problem is computationally difficult to solve; therefore, the principle objective in the design of GRAM is to facilitate man-machine collaborative problem solving. GRAM quickly approximates a solution, as follows. (i) A clustering algorithm determines a probable set of restriction fragments. (ii) An assembly algorithm permutes the set of restriction fragments such that the maximal number of clone fragments are contiguous. The output of the GRAM algorithm is displayed for the user to query and edit. This paper describes the stochastic assembly algorithm and shows how it works with the interactive graphics to support man-machine problem solving. In order to test and verify the performance of GRAM, we have developed a program called genfragII to simulate the digestion of clones and fragments; this program is described and results are presented. GRAM is also being used for a number of genome mapping projects.

Integration of competing ancillary assertions in genome assembly.

Assembly of genomic sequences and maps relies on a primary set of experimental data (e.g., the sequences of individual DNA fragments, or hybridization fingerprints of individual clone inserts), but almost always also relies on several streams of related but distinct kinds of data for completeness and accuracy of the final construction. These secondary data sets, which we term ancillary information, usually contain errors (as do the primary data sets, therefore creating the possibility of conflict between data sets), often arise from different experimental protocols and correspond to different scales of measurement, and occasionally include non-quantitative statements about the data. We present an approach for integration of ancillary assertions in the optimization of genome assembly, based on simultaneous balancing among the primary and secondary data sets, and include specific examples in the context of assembling DNA sequencing fragments to reconstruct a parent sequence.

The density of transcriptional elements in promoter and non-promoter sequences.

Using data currently available from transcriptional element (TE) databases, we have analyzed the density of these elements in both promoter and non-promoter sequences obtained from the GenBank sequence database. The density of putative TEs in non-promoter sequences was about 16% higher than that seen in pseudo-random DNA sequences. Promoter TE density from the transcription startsite to 100 basepairs upstream was found to be about 42% higher than in non-promoter sequences. However, the extensive overlap of putative TE densities between promoters and non-promoters confounds an attempt to use TE density as a simple discriminator.

Artificially generated data sets for testing DNA sequence assembly algorithms.

We have developed a set of tools, genfrag, to fragment and optionally mutate a DNA sequence to generate benchmark data sets for testing DNA sequence assembly algorithms. Data parameters can be systematically and independently varied to explore the range of data--and corresponding performance of assembly tools--encountered on large-scale random, or "shot-gun," sequencing projects.

Genetic algorithms for DNA sequence assembly.

This paper describes a genetic algorithm application to the DNA sequence assembly problem. The genetic algorithm uses a sorted order representation for representing the orderings of fragments. Two different fitness functions, both based on pairwise overlap strengths between fragments, are tested. The paper concludes that the genetic algorithm is a promising method for fragment assembly problems, achieving usable solutions quickly, but that the current fitness functions are flawed and that other representations might be more appropriate.

Splicing signals in Drosophila: intron size, information content, and consensus sequences.

A database of 209 Drosophila introns was extracted from Genbank (release number 64.0) and examined by a number of methods in order to characterize features that might serve as signals for messenger RNA splicing. A tight distribution of sizes was observed: while the smallest introns in the database are 51 nucleotides, more than half are less than 80 nucleotides in length, and most of these have lengths in the range of 59-67 nucleotides. Drosophila splice sites found in large and small introns differ in only minor ways from each other and from those found in vertebrate introns. However, larger introns have greater pyrimidine-richness in the region between 11 and 21 nucleotides upstream of 3' splice sites. The Drosophila branchpoint consensus matrix resembles C T A A T (in which branch formation occurs at the underlined A), and differs from the corresponding mammalian signal in the absence of G at the position immediately preceding the branchpoint. The distribution of occurrences of this sequence suggests a minimum distance between 5' splice sites and branchpoints of about 38 nucleotides, and a minimum distance between 3' splice sites and branchpoints of 15 nucleotides. The methods we have used detect no information in exon sequences other than in the few nucleotides immediately adjacent to the splice sites. However, Drosophila resembles many other species in that there is a discontinuity in A + T content between exons and introns, which are A + T rich.

GenBank.

The GenBank nucleotide sequence database now contains sequence data and associated annotation corresponding to 85,000,000 nucleotides in 67,000 entries from a total of 3,000 organisms. The input stream of data coming into the database is primarily as direct submissions from the scientific community on electronic media, with little or no data being keyboarded from the printed page by the databank staff. The data are maintained in a relational database management system and are made available in flatfile form through on-line access, and through various network and off-line computer-readable media. The data are also distributed in relational form through satellite copies at a number of institutions in the U.S. and elsewhere. In addition, GenBank provides the U.S. distribution center for the BIOSCI electronic bulletin board service.

Identifying potential tRNA genes in genomic DNA sequences.

We have developed an algorithm that automatically and reproducibly identifies potential tRNA genes in genomic DNA sequences, and we present a general strategy for testing the sensitivity of such algorithms. This algorithm is useful for the flagging and characterization of long genomic sequences that have not been experimentally analyzed for identification of functional regions, and for the scanning of nucleotide sequence databases for errors in the sequences and the functional assignments associated with them. In an exhaustive scan of the GenBank database, 97.5% of the 744 known tRNA genes were correctly identified (true-positives), and 42 previously unidentified sequences were predicted to be tRNAs. A detailed analysis of these latter predictions reveals that 16 of the 42 are very similar to known tRNA genes, and we predict that they do, in fact, code for tRNA, yielding a false-positive rate for the algorithm of 0.003%. The new algorithm and testing strategy are a considerable improvement over any previously described strategies for recognizing tRNA genes, and they allow detections of genes (including introns) embedded in long genomic sequences.

A program for computer-assisted scoring of Southern blots.

SCORE, a program for computer-assisted scoring of Southern blots of clone DNA, retains the use of expert human judgment while taking over much of the drudgery of the scoring task. The primary functions of the program are to help make an aligned overlay of the fluorescence gel image and the autoradiogram blot image, to keep track of band and lane locations and to store the resulting data directly into a database. Use of SCORE has resulted in greatly increased efficiency and accuracy.

Electronic data publishing and GenBank.

GenBank, the national repository for nucleotide sequence data, has implemented a new model of scientific data management, which we term electronic data publishing. In traditional publishing, both scientific conclusions and supporting data are communicated via the printed page, and in electronic journal publishing, both types of information are communicated via electronic media. In electronic data publishing, by contrast, conclusions are published in a journal while data are published via a network-accessible, electronic database.