Microarray-based capture of novel expressed cell type-specific transfrags (CoNECT) to annotate tissue-specific transcription in Drosophila melanogaster.

Faithful annotation of tissue-specific transcript isoforms is important not only to understand how genes are organized and regulated but also to identify potential novel, unannotated exons of genes, which may be additional targets of mutation in disease states or while performing mutagenic screens. We have developed a microarray enrichment methodology followed by long-read, next-generation sequencing for identification of unannotated transcript isoforms expressed in two Drosophila tissues, the ovary and the testis. Even with limited sequencing, these studies have identified a large number of novel transcription units, including 5' exons and extensions, 3' exons and extensions, internal exons and exon extensions, gene fusions, and both germline-specific splicing events and promoters. Additionally, comparing our capture dataset with tiling array and traditional RNA-seq analysis, we demonstrate that our enrichment strategy is able to capture low-abundance transcripts that cannot readily be identified by the other strategies. Finally, we show that our methodology can help identify transcriptional signatures of minority cell types within the ovary that would otherwise be difficult to reveal without the CoNECT enrichment strategy. These studies introduce an efficient methodology for cataloging tissue-specific transcriptomes in which specific classes of genes or transcripts can be targeted for capture and sequence, thus reducing the significant sequencing depth normally required for accurate annotation. Ovary and testis isotigs over 200 bp have been deposited with the GenBank Transcriptome Shotgun Assembly Sequence Database as bioproject no.PRJNA89451 (accession nos. JV208106­JV230865).

Diversity of conotoxin types from Conus californicus reflects a diversity of prey types and a novel evolutionary history.

Most species within the genus Conus are considered to be specialists in their consumption of prey, typically feeding on molluscs, vermiform invertebrates or fish, and employ peptide toxins to immobilize prey. Conus californicus Hinds 1844 atypically utilizes a wide range of food sources from all three groups. Using DNA- and protein-based methods, we analyzed the molecular diversity of C. californicus toxins and detected a correspondingly large number of conotoxin types. We identified cDNAs corresponding to seven known cysteine-frameworks containing over 40 individual inferred peptides. Additionally, we found a new framework (22) with six predicted peptide examples, along with two forms of a new peptide type of unusual length. Analysis of leader sequences allowed assignment to known superfamilies in only half of the cases, and several of these showed a framework that was not in congruence with the identified superfamily. Mass spectrometric examination of chromatographic fractions from whole venom served to identify peptides corresponding to a number of cDNAs, in several cases differing in their degree of posttranslational modification. This suggests differential or incomplete biochemical processing of these peptides. In general, it is difficult to fit conotoxins from C. californicus into established toxin classification schemes. We hypothesize that the novel structural modifications of individual peptides and their encoding genes reflect evolutionary adaptation to prey species of an unusually wide range as well as the large phylogenetic distance between C. californicus and Indo-Pacific species.

A diverse family of novel peptide toxins from an unusual cone snail, Conus californicus.

Diversity among Conus toxins mirrors the high species diversity in the Indo-Pacific region, and evolution of both is thought to stem from feeding-niche specialization derived from intra-generic competition. This study focuses on Conus californicus, a phylogenetic outlier endemic to the temperate northeast Pacific. Essentially free of congeneric competitors, it preys on a wider variety of organisms than any other cone snail. Using molecular cloning of cDNAs and mass spectrometry, we examined peptides isolated from venom ducts to elucidate the sequences and post-translational modifications of two eight-cysteine toxins (cal12a and cal12b of type 12 framework) that block voltage-gated Na(+) channels. Based on homology of leader sequence and mode of action, these toxins are related to the O-superfamily, but differ significantly from other members of that group. Six of the eight cysteine residues constitute the canonical framework of O-members, but two additional cysteine residues in the N-terminal region define an O+2 classification within the O-superfamily. Fifteen putative variants of Cal12.1 toxins have been identified by mRNAs that differ primarily in two short hypervariable regions and have been grouped into three subtypes (Cal12.1.1-3). This unique modular variation has not been described for other Conus toxins and suggests recombination as a diversity-generating mechanism. We propose that these toxin isoforms show specificity for similar molecular targets (Na(+) channels) in the many species preyed on by C. californicus and that individualistic utilization of specific toxin isoforms may involve control of gene expression.

Ultra-high resolution array painting facilitates breakpoint sequencing.

OBJECTIVE: To describe a considerably advanced method of array painting, which allows the rapid, ultra-high resolution mapping of translocation breakpoints such that rearrangement junction fragments can be amplified directly and sequenced. METHOD: Ultra-high resolution array painting involves the hybridisation of probes generated by the amplification of small numbers of flow-sorted derivative chromosomes to oligonucleotide arrays designed to tile breakpoint regions at extremely high resolution. RESULTS AND DISCUSSION: How ultra-high resolution array painting of four balanced translocation cases rapidly and efficiently maps breakpoints to a point where junction fragments can be amplified easily and sequenced is demonstrated. With this new development, breakpoints can be mapped using just two array experiments: the first using whole-genome array painting to tiling resolution large insert clone arrays, the second using ultra-high-resolution oligonucleotide arrays targeted to the breakpoint regions. In this way, breakpoints can be mapped and then sequenced in a few weeks.

Integrative approaches to determining Csl function.

While there is an ever-increasing amount of information regarding cellulose synthase catalytic subunits (CesA) and their role in the formation of the cell wall, the remainder of the enzymes that synthesize structural cell wall polysaccharides are unknown. The completion of the Arabidopsis genome and the wealth of the sequence information from other plant genome projects provide a rich resource for determining the identity of these enzymes. Arabidopsis contains six families of genes related to cellulose synthase, the cellulose synthase-like (Csl) genes. Our laboratory is taking a multidisciplinary approach to determine the function of the Csl genes, incorporating genomic, genetic and biochemical data. Information from expressed sequence tag (EST) projects has revealed the presence of Csl genes in all plant species with a significant number of ESTs. Certain Csl families appear to be missing from some species. For example, no examples of CslG ESTs have been found in rice or maize. Microarray data and reporter constructs are being used to determine the expression pattern of the CesA and Csl genes in Arabidopsis. Mutations and insertion events have been identified in a majority of the genes in the Arabidopsis CesA superfamily and are being characterized by phenotypic and biochemical analysis. While we cannot yet link the function of any of the Csl genes to their respective products, the expression and localization of these genes is consistent with the expected expression pattern of polysaccharide synthases that contribute to the primary cell wall.

Engineered metal binding sites on green fluorescence protein.

The ability to assay a variety of metals by noninvasive methods has applications in both biomedical and environmental research. Green fluorescent protein (GFP) is a protein isolated from coelenterates that exhibits spontaneous fluorescence. GFP does not require any exogenous cofactors for fluorescence, and can be easily appended to other proteins at the DNA level, producing a fluorescence-labeled target protein in vivo. Metals in close proximity to chromophores are known to quench fluorescence in a distance-dependent fashion. Potential metal binding sites on the surface of GFP have been identified and mutant proteins have been designed, created, and characterized. These metal-binding mutants of GFP exhibit fluorescence quenching at lower transition metal ion concentrations than those of the wild-type protein. These GFP mutants represent a new class of protein-based metal sensors.

A defect in beta-oxidation causes abnormal inflorescence development in Arabidopsis.

The abnormal inflorescence meristem1 (aim1) mutation affects inflorescence and floral development in Arabidopsis. After the transition to reproductive growth, the aim1 inflorescence meristem becomes disorganized, producing abnormal floral meristems and resulting in plants with severely reduced fertility. The derived amino acid sequence of AIM1 shows extensive similarity to the cucumber multifunctional protein involved in beta-oxidation of fatty acids, which possesses l-3-hydroxyacyl-CoA hydrolyase, l-3-hydroxyacyl-dehydrogenase, d-3-hydroxyacyl-CoA epimerase, and Delta(3), Delta(2)-enoyl-CoA isomerase activities. A defect in beta-oxidation has been confirmed by demonstrating the resistance of the aim1 mutant to 2,4-diphenoxybutyric acid, which is converted to the herbicide 2,4-D by the beta-oxidation pathway. In addition, the loss of AIM1 alters the fatty acid composition of the mature adult plant.

The fate of inflorescence meristems is controlled by developing fruits in Arabidopsis.

The relationship between fruit development and the proliferative capacities of inflorescence meristems has been examined in Arabidopsis thaliana. In the wild-type Landsberg erecta (Ler) line, flower production ceases coordinately on all inflorescence branches by a process we have designated global proliferative arrest (GPA). Morphological studies indicate that GPA involves a cessation of proliferative activity at the meristems, but a retention of the structural characteristics of the proliferating meristems. GPA does not occur in the male-sterile (ms1-1) line, nor in wild-type Ler when fruits are surgically removed. In these cases, inflorescence meristems continue to proliferate, ultimately terminating by a different process, designated terminal differentiation, in which disruptions in patterning at the apex are followed by the loss of the inflorescence meristem. We present an argument that GPA is mediated by a specific communication system between inflorescence meristems and developing fruits. Analysis of reduced-fertility mutants provided evidence that GPA is dependent on seed development specifically. Mutations conferring hormone deficiency or insensitivity did not disrupt the correlative interactions leading to GPA.