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Stylonychia lemnae is a classical model single-celled eukaryote, and a quintessential ciliate typified by dimorphic nuclei: A small, germline micronucleus and a massive, vegetative macronucleus. The genome within Stylonychia's macronucleus has a very unusual architecture, comprised variably and highly amplified "nanochromosomes," each usually encoding a single gene with a minimal amount of surrounding noncoding DNA. As only a tiny fraction of the Stylonychia genes has been sequenced, and to promote research using this organism, we sequenced its macronuclear genome. We report the analysis of the 50.2-Mb draft S. lemnae macronuclear genome assembly, containing in excess of 16,000 complete nanochromosomes, assembled as less than 20,000 contigs. We found considerable conservation of fundamental genomic properties between S. lemnae and its close relative, Oxytricha trifallax, including nanochromosomal gene synteny, alternative fragmentation, and copy number. Protein domain searches in Stylonychia revealed two new telomere-binding protein homologs and the presence of linker histones. Among the diverse histone variants of S. lemnae and O. trifallax, we found divergent, coexpressed variants corresponding to four of the five core nucleosomal proteins (H1.2, H2A.6, H2B.4, and H3.7) suggesting that these ciliates may possess specialized nucleosomes involved in genome processing during nuclear differentiation. The assembly of the S. lemnae macronuclear genome demonstrates that largely complete, well-assembled highly fragmented genomes of similar size and complexity may be produced from one library and lane of Illumina HiSeq 2000 shotgun sequencing. The provision of the S. lemnae macronuclear genome sets the stage for future detailed experimental studies of chromatin-mediated, RNA-guided developmental genome rearrangements.

A model for integrating course-based research with community genome annotation efforts at model organism databases is presented. Disseminating gene function discoveries directly to an interested audience increased student motivation to more deeply engage all aspects of an authentic research experience., Use of inquiry-based research modules in the classroom has soared over recent years, largely in response to national calls for teaching that provides experience with scientific processes and methodologies. To increase the visibility of in-class studies among interested researchers and to strengthen their impact on student learning, we have extended the typical model of inquiry-based labs to include a means for targeted dissemination of student-generated discoveries. This initiative required: 1) creating a set of research-based lab activities with the potential to yield results that a particular scientific community would find useful and 2) developing a means for immediate sharing of student-generated results. Working toward these goals, we designed guides for course-based research aimed to fulfill the need for functional annotation of the Tetrahymena thermophila genome, and developed an interactive Web database that links directly to the official Tetrahymena Genome Database for immediate, targeted dissemination of student discoveries. This combination of research via the course modules and the opportunity for students to immediately “publish” their novel results on a Web database actively used by outside scientists culminated in a motivational tool that enhanced students’ efforts to engage the scientific process and pursue additional research opportunities beyond the course.

As the world’s population grows, access to a safe food supply will continue to be a global priority. In recent years, the world has experienced an increase in mycotoxin contamination of grains due to climatic and agronomic changes that encourage fungal growth during cultivation. A number of the molds that are plant pathogens produce trichothecene mycotoxins, which are known to cause serious human and animal toxicoses. This review covers the types of trichothecenes, their complexity, and proposed biosynthetic pathways of trichothecenes.

We have discovered a three-gene macronuclear chromosome in a spirotrichous ciliate of the genus Holosticha. From 5' to 3', this chromosome contains genes encoding a member of the small G-protein family, an NAD kinase domain-containing protein, and the large subunit of DNA polymerase alpha. These three genes are separated by 16 and 38 nucleotides, respectively, and are oriented in the same direction in both the macronuclear and the micronuclear genomes. Probes made to these genes all hybridize to a single, strong band of size 7.0 kbp on a Southern Blot of Holosticha sp. macronuclear DNA, corresponding to the size of the three-gene macronuclear chromosome. Mapping the 5' and 3' ends of each of these genes using RACE showed that the transcripts of these genes exist as discrete mRNAs that are capped and polyadenylated. No nucleotides appeared to be added at the 5' ends of these transcripts, indicating that these transcripts are not generated by alternative or trans-splicing, but rather that each gene is transcribed from its own distinct promoter. Analysis of these linked genes may help define the evolutionary pressures leading to the extensive chromosome fragmentation seen in spirotrichous ciliates.

Ciliates, microbial eukaryotes, have been important experimental organisms for >100 years, contributing to the discovery and understanding of self-splicing RNA, telomere biochemistry and transcriptional regulation by histone modification. We have completed a pilot sequencing project of the Oxytricha trifallax macronuclear (MAC) genome. When complete, this genome sequence, along with the emerging Tetrahymena thermophila and Paramecium tetraurelia sequences, will provide an important resource for basic eukaryotic cell biology, molecular evolution and the related apicomplexan parasites, such as Plasmodium spp.

BLAST is the most widely used software in bioinformatics research. Its main function is to compare a sequence of interest, the query sequence, to sequences in a large database. BLAST then reports the best matches, or “hits,” found in the database. This simple program has two primary applications. First, if the function of the query sequence is unknown, it may be possible to infer its function based on the recognized functions of similar sequences. Second, if the researcher has a query sequence with a known function, it may be possible to identify sequences in the database that have similar functions. The utility of BLAST therefore depends on the researcher's choice of query sequence and database. An appreciation for the functions and limitations of BLAST are vital to using this program effectively. This unit will introduce the basic concepts behind BLAST, walk through BLAST searching protocols, and interpret common results. Curr. Protoc. Essential Lab. Tech. 1:11.1.1-11.1.36. © 2009 by John Wiley & Sons, Inc. Keywords: BLAST; sequence alignment; sequence analysis; sequence annotation

The surprising discovery of a whole-genome duplication in the otherwise compact genome of Paramecium tetraurelia displays the early forces driving gene retention and loss.

Several studies have provided strong, but indirect evidence that signalling through pathways involving protein kinase C (PKC) plays an important role in morphogenesis and patterning in Hydra. We have cloned a gene (HvPKC2) from Hydra vulgaris which encodes a member of the nPKC subfamily. In adult polyps, HvPKC2 is expressed at high levels in two locations, the endoderm of the foot and the endoderm of the hypostomal tip. Increased expression of HvPKC2 is an early event during head and foot regeneration, with the rise in expression being restricted to the endodermal cells underlying the regenerating ends. No upregulation is observed if regenerates are cut too close to the head to form a foot. Elevated expression of HvPKC2 is also observed in the endoderm underlying lithium-induced ectopic feet. A dynamic and complex pattern of expression is seen in developing buds. Regeneration of either head or foot is accompanied by an increase in the amount of PKC in both soluble and particulate fractions. An increase in the fraction of PKC activity which is membrane-bound is specifically associated with head regeneration. Taken together these data suggest that patterning of the head and foot in Hydra is controlled in part by the level of HvPKC2 expression, whilst head formation is accompanied by an in vivo activation of both calcium-dependent and independent PKC isoforms.

With more chromosomes than any other sequenced genome, the macronuclear genome of Oxytricha trifallax has a unique and complex architecture, including alternative fragmentation and predominantly single-gene chromosomes., The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor “silent” germline micronuclear genome by a process of “unscrambling” and fragmentation. The tiny macronuclear “nanochromosomes” typically encode single, protein-coding genes (a small portion, 10%, encode 2–8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease., Author Summary The macronuclear genome of the ciliate Oxytricha trifallax, contained in its somatic nucleus, has a unique genome architecture. Unlike its diploid germline genome, which is transcriptionally inactive during normal cellular growth, the macronuclear genome is fragmented into at least 16,000 tiny (∼3.2 kb mean length) chromosomes, most of which encode single actively transcribed genes and are differentially amplified to a few thousand copies each. The smallest chromosome is just 469 bp, while the largest is 66 kb and encodes a single enormous protein. We found considerable variation in the genome, including frequent alternative fragmentation patterns, generating chromosome isoforms with shared sequence. We also found limited variation in chromosome amplification levels, though insufficient to explain mRNA transcript level variation. Another remarkable feature of Oxytricha's macronuclear genome is its inordinate fondness for telomeres. In conjunction with its possession of tens of millions of chromosome-ending telomeres per macronucleus, we show that Oxytricha has evolved multiple putative telomere-binding proteins. In addition, we identified two new domesticated transposase-like protein classes that we propose may participate in the process of genome rearrangement. The macronuclear genome now provides a crucial resource for ongoing studies of genome rearrangement processes that use Oxytricha as an experimental or comparative model.

Within the past decade, genomic studies have emerged as essential and highly productive tools to explore the biology of Tetrahymena thermophila. The current major resources, which have been extensively mined by the research community, are the annotated macronuclear genome assembly, transcriptomic data and the databases that house this information. Efforts in progress will soon improve these data sources and expand their scope, including providing annotated micronuclear and comparative genomic sequences. Future studies of Tetrahymena cell and molecular biology, development, physiology, evolution and ecology will benefit greatly from these resources and the advanced genomic technologies they enable.

When funding for Tetrahymena Genome Database (TGD) ended in 2006, no further updates were made to this important community resource and the main database was taken offline in 2008. We have restored and updated this important resource for use by the Tetrahymena research community. We have also retooled the TGD website (now TGD Wiki) to allow members of the community to directly update the information presented for each gene, including gene names, descriptions and Gene Ontology annotations, from a web browser. Maintenance of genome annotations by the authors generating and publishing primary data, rather than dedicated scientific curators, is a viable alternative for the upkeep of genomes, particularly for organisms with smaller research communities. By combining simple, intuitive displays with the powerful search functions made possible by its underlying relational database, TGD Wiki has been designed to maximize participation by bench scientists in the development of their community bioinformatics resource. Database URL: http://ciliate.org

Within the past decade, genomic studies have emerged as essential and highly productive tools to explore the biology of Tetrahymena thermophila. The current major resources, which have been extensively mined by the research community, are the annotated macronuclear genome assembly, transcriptomic data and the databases that house this information. Efforts in progress will soon improve these data sources and expand their scope, including providing annotated micronuclear and comparative genomic sequences. Future studies of Tetrahymena cell and molecular biology, development, physiology, evolution and ecology will benefit greatly from these resources and the advanced genomic technologies they enable.

Background Fused genes are important sources of data for studies of evolution and protein function. To date no service has been made available online to aid in the large-scale identification of fused genes in sequenced genomes. We have developed a program, Gene deFuser, that analyzes uploaded protein sequence files for characteristics of gene fusion events and presents the results in a convenient web interface. Results To test the ability of this software to detect fusions on a genome-wide scale, we analyzed the 24,725 gene models predicted for the ciliated protozoan Tetrahymena thermophila. Gene deFuser detected members of eight of the nine families of gene fusions known or predicted in this species and identified nineteen new families of fused genes, each containing between one and twelve members. In addition to these genuine fusions, Gene deFuser also detected a particular type of gene misannotation, in which two independent genes were predicted as a single transcript by gene annotation tools. Twenty-nine of the artifacts detected by Gene deFuser in the initial annotation have been corrected in subsequent versions, with a total of 25 annotation artifacts (about 1/3 of the total fusions identified) remaining in the most recent annotation. Conclusions The newly identified Tetrahymena fusions belong to classes of genes involved in processes such as phospholipid synthesis, nuclear export, and surface antigen generation. These results highlight the potential of Gene deFuser to reveal a large number of novel fused genes in evolutionarily isolated organisms. Gene deFuser may also prove useful as an ancillary tool for detecting fusion artifacts during gene model annotation.

Fusions of the first two enzymes in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconolactonase (6PGL), have been previously described in two distant clades, chordates and species of the malarial parasite Plasmodium. We have analyzed genome and expressed sequence data from a variety of organisms to identify the origins of these gene fusion events. Based on the orientation of the domains and range of species in which homologs can be found, the fusions appear to have occurred independently, near the base of the metazoan and apicomplexan lineages. Only one of the two metazoan paralogs of G6PD is fused, showing that the fusion occurred after a duplication event, which we have traced back to an ancestor of choanoflagellates and metazoans. The Plasmodium genes are known to contain a functionally important insertion that is not seen in the other apicomplexan fusions, highlighting this as a unique characteristic of this group. Surprisingly, our search revealed two additional fusion events, one that combined 6PGL and G6PD in an ancestor of the protozoan parasites Trichomonas and Giardia, and another fusing G6PD with phosphogluconate dehydrogenase (6PGD) in a species of diatoms. This study extends the range of species known to contain fusions in the pentose phosphate pathway to many new medically and economically important organisms.

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.

Spliced leader trans-splicing is an mRNA maturation process used by a small set of eukaryotes, including the nematode C. elegans, to cap the downstream genes of operons. We analyzed the frequency of duplication of operonic genes in C. elegans and confirmed that they are duplicated less often in the genome than monocistronic genes. Because operons account for about 15% of the genes in C. elegans, this lower duplication frequency might place a large constraint on the plasticity of the genome. Further analyses suggest that this paucity of duplicated genes results from operon organization hindering specific types of gene duplication.

During the course of a pilot genome project for the ciliate Oxytricha trifallax, we discovered a fusion gene never before described in any taxa. This gene, FSF1, encodes a putative fusion protein comprising an entire formaldehyde dehydrogenase (FALDH) homolog at one end and an S-formylglutathione hydrolase (SFGH) homolog at the other, two proteins that catalyze serial steps in the formaldehyde detoxification pathway. We confirmed the presence of the Oxytricha fusion gene in vivo and detected transcripts of the full-length fusion gene. A survey of other large-scale sequencing projects revealed a similar fusion protein in a distantly related ciliate, Tetrahymena thermophila, and a possible fusion of these two genes in the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana, but in the reverse order, with the SFGH domain encoded upstream of the FALDH domain. Orthologs of these fusion proteins may be widespread within the ciliates and diatoms.

The macronuclear genomes of spirotrichous ciliates are almost entirely polyploid, single-gene chromosomes ("nanochromosomes"). We recently performed a pilot genome project for a member of this group, Oxytricha trifallax ( Sterkiella histriomuscorum), in which approximately 2000 nanochromosomes were cloned at random and end-sequenced. Here we describe the global properties of the coding regions predicted for these molecules, including nucleotide composition, codon usage, and intron properties. In identifying splice donor, acceptor and branch sites, we found that longer introns in Oxytricha have a stronger signal at the donor site than do smaller introns, as has been found for Caenorhabditis elegans and Drosophila, despite the overall small size of the introns. A systematic search for multi-gene chromosomes identified 11 candidate nanochromosomes. We compare the results from this large dataset with those obtained from earlier studies and with statistics recorded from ciliates and other eukaryotes.

A search of databases with the sequence from the 5′ untranslated region of a Hydra cDNA clone encoding a receptor protein-tyrosine kinase revealed that a number of Hydra cDNAs contain one of two different sequences at their 5′ ends. This finding suggested the possibility that mRNAs in Hydra receive leader sequences by trans - splicing. This hypothesis was confirmed by the finding that the leader sequences are transcribed as parts of small RNAs encoded by genes located in the 5S rRNA clusters of Hydra . The two spliced leader (SL) RNAs (SL-A and -B) contain splice donor dinucleotides at the predicted positions, and genes that receive SLs contain splice acceptor dinucleotides at the predicted positions. Both of the SL RNAs are bound by antibody against trimethylguanosine, suggesting that they contain a trimethylguanosine cap. The predicted secondary structures of the Hydra SL RNAs show significant differences from the structures predicted for the SLs of other organisms. Messenger RNAs have been identified that can receive either SL-A or -B, although the impact of the two different SLs on the function of the mRNA is unknown. The presence and features of SL addition in the phylum Cnidaria raise interesting questions regarding the evolution of this process.

Syk family protein-tyrosine kinases are essential components of immunoreceptor signaling in mammalian lymphocytes. The absence of Syk genes from the Caenorhabditis elegans genome suggests that this kinase family is of recent evolutionary origin. Surprisingly, we have found that Hydra vulgaris, a member of the early diverging animal phylum Cnidaria, contains a gene encoding a Syk kinase. Phylogenetic analysis indicates that a single Syk family gene was present in animals prior to the gene duplication that gave rise to Syk and ZAP-70, the two mammalian Syk family genes. C. elegans also lacks a Shark protein-tyrosine kinase gene, which we show is a member of a sister group to the Syk family. We conclude that both Syk and Shark genes were lost from the genome of an ancestor of C. elegans. This natural gene knockout result indicates that neither Syk nor Shark kinases are essential for processes held in common between the nematode and other metazoans. The Hydra Syk gene is expressed in epithelial cells, a site consistent with a role for Hydra Syk in recognition of foreign cells.

What is spliced leader (SL) trans-splicing? It is an mRNA maturation process, similar to intron splicing, which has been shown to occur in a limited number of eukaryotes. In SL trans-splicing, the cell replaces nucleotides at the 5′ end of some pre-mRNAs with those of a special class of small nuclear RNAs, called SL RNAs. These are short molecules with two functionally distinct halves: the 5′ half consists of the leader sequence that is transferred to a pre-mRNA, along with the SL RNA's methylguanosine cap; the 3′ half contains a binding site for the Sm protein complex, which binds many of the RNAs involved in intron splicing. These two halves are separated by a splice donor site, a GT dinucleotide. Nuclear machinery trans-splices the leader sequence to splice acceptor sites (AG dinucleotides) in the 5′ region of target pre-mRNAs. As a result, many mRNAs in SL trans-splicing species have a common sequence at the 5′ end.How is SL trans-splicing related to intron splicing? The mechanism of SL trans-splicing is very similar to cis- (intron) splicing (Figure 1Figure 1). In both cases, the 2′ hydroxyl group of a nucleotide (usually adenosine) severs the pre-mRNA backbone at the splice donor site, freeing the upstream exon to displace the intron sequence at the splice acceptor. In intron splicing, the splice donor and acceptor sites lie on the same strand of RNA, separated by the intron sequence, which contains the branch point adenosine. For SL trans-splicing, the splice donor site of the SL RNA is attacked by an adenosine between the 5′ end of the pre-mRNA and its SL addition site. The region of the pre-mRNA upstream of the SL addition site, which is removed when the leader sequence is attached, is known as the ‘outron’ — it is ‘outside’ the gene, whereas introns are ‘inside’.Figure 1The leader sequence (yellow box) of an SL RNA is attached to the first exon (orange box) of a pre-mRNA by a trans-splicing reaction.The outron of the pre-mRNA and the intron-like portion of the SL RNA form a ‘Y’ branched byproduct, similar to the lariat structure formed during intron splicing. The 2,2,7-trimethylguanosine cap structure (or ‘Cap 4’ in trypanosomes) found on SL RNAs and trans-spliced mRNAs (blue circles) differs from the 7-methylguanosine caps typically found on mRNAs (red circles). These caps impart novel properties to trans-spliced messages.View Large Image | View Hi-Res Image | Download PowerPoint SlideStrikingly, experiments in the nematode Caenorhabditis elegans demonstrated that the relative location of the donor and acceptor sites is sufficient to determine whether a splice acceptor is cis- or trans-spliced. Cis-spliced acceptors can be converted to trans-splicing acceptors if the donor site upstream is mutated, and similarly an SL addition site will be cis-spliced if a donor site is inserted upstream.What is the function of SL trans-splicing? SL addition provides the cell with an alternative way of capping mRNAs, a modification required for mRNA stability, transport and translation. The standard capping machinery is typically recruited by RNA polymerase II at the beginning of transcription to cap the growing RNA. In trypanosomes, SL addition is used to cap a subset of pre-mRNAs transcribed by RNA polymerase I, which does not recruit the capping machinery. In a wider range of eukaryotes, SL addition allows the formation of operons — adjacent genes that are transcribed as a single primary transcript. The cleavage reaction that precedes polyadenylation of each gene effectively chops the transcript into smaller pieces. Since only the message from the 5′ end retains the transcript's original cap, SL trans-splicing is needed to cap the remaining fragments.Addition of an SL has also been shown to affect the translation rate of some genes, add missing start codons and trim off outron sequences. But while the role of SL addition in these processes is well established, the benefit of transcription by polymerase I, inclusion in an operon, translational regulation, substitution of a start codon and outron removal is not immediately obvious for most trans-spliced genes.How is it phylogenetically distributed? The complete phylogenetic distribution of SL trans-splicing is not currently known. To date, SL trans-splicing has been found in six diverse groups of eukaryotes: nematodes, flatworms, cnidarians, ascidians, rotifers and euglenozoans. But it has not been detected in other well-studied eukaryotic taxa, such as fungi, plants, vertebrates and arthropods.How did it evolve? There are two competing hypotheses describing the origin of SL trans-splicing. The ‘SL trans-splicing early’ hypothesis proposes that SL trans-splicing was present in the ancestral eukaryote and subsequently lost in most phyla. This hypothesis is supported in part by the continuing discovery of SL trans-splicing in an expanding range of eukaryotes. The ‘SL trans-splicing late’ hypothesis proposes that the process has emerged several times independently, and that features unique to SL addition — the SL RNA, trans-spliceosome-specific proteins, SL-specific translation enhancer proteins, operons, and so on — are also independently derived in these lineages. This hypothesis is supported by the observation that the few unique components shown to be involved in SL addition are not obviously conserved across different trans-splicing phyla. Further studies into the mechanism and effects of SL trans-splicing in different phyla will help to clarify its origin.Why should I care? Many of the organisms that perform SL trans-splicing, such as trypanosomes, flatworms and nematodes are pathogenic to humans. Drugs that target components of the trans-spliceosome or disrupt the downstream effects of SL trans-splicing may be highly effective against these parasitic species, while causing little harm to patients.

Axial patterning of the aboral end of the hydra body column was examined using expression data from two genes. One, shin guard, is a novel receptor protein-tyrosine kinase gene expressed in the ectoderm of the peduncle, the end of the body column adjacent to the basal disk. The other gene, manacle, is a paired-like homeobox gene expressed in differentiating basal disk ectoderm. During regeneration of the aboral end, expression of manacle precedes that of shin guard. This result is consistent with a requirement for induction of peduncle tissue by basal disk tissue. Our data contrast with data on regeneration of the oral end. During oral end regeneration, markers for tissue of the tentacles, which lie below the extreme oral end (the hypostome), are detected first. Later, markers for the hypostome itself appear at the regenerating tip, with tentacle markers displaced to the region below. Additional evidence that tissue can form basal disk without passing through a stage as peduncle tissue comes from LiCl-induced formation of patches of ectopic basal disk tissue. While manacle is ectopically expressed during formation of basal disk patches, shin guard is not. The genes examined also provide new information on development of the aboral end in buds. Although adult hydra are radially symmetrical, expression of both genes in the bud's aboral end is initially asymmetrical, appearing first on the side of the bud closest to the parent's basal disk. The asymmetry can be explained by differences in positional information in the body column tissue that evaginates to form a bud. As predicted by this hypothesis, grafts reversing the orientation of evaginating body column tissue also reverse the orientation of asymmetrical gene expression.

The Hydra EST project is being carried out in collaboration with the Genome Sequencing Center at Washington University. The project is generously supported by grant IBN-0120591 from the National Science Foundation.