The dwarfin protein family has been genetically implicated in transforming growth factor beta (TGF-beta)-like signaling pathways in Drosophila and Caenorhabditis elegans. To investigate the role of these proteins in mammalian signaling pathways, we have isolated and studied two murine dwarfins, dwarfin-A and dwarfin-C. Using antibodies against dwarfin-A and dwarfin-C, we show that these two dwarfins and an immunogenically related protein, presumably also a dwarfin, are phosphorylated in a time- and dose-dependent manner in response to TGF-beta. Bone morphogenetic protein 2, a TGF-beta superfamily ligand, induces phosphorylation of only the related dwarfin protein. Thus, TGF-beta superfamily members may use overlapping yet distinct dwarfins to mediate their intracellular signals. Furthermore, transient overexpression of either dwarfin-A or dwarfin-C causes growth arrest, implicating the dwarfins in growth regulation. This work provides strong biochemical and preliminary functional evidence that dwarfin-A and dwarfin-C represent prototypic members of a family of mammalian proteins that may serve as mediators of signaling pathways for TGF-beta superfamily members.

TGF-beta-related signaling pathways play diverse roles during vertebrate and invertebrate development. A common mechanism for regulating the activity of TGF-beta family members is inhibition by extracellular antagonists. Recently, the Drosophila short gastrulation (sog) gene was shown to encode a predicted diffusible factor which antagonizes signaling mediated by the TGF-beta-like Decapentaplegic (Dpp) pathway in the early blastoderm embryo. sog and dpp, which are among the earliest zygotic genes to be activated, are expressed in complementary dorsal-ventral domains. The opposing actions of sog and dpp in the early embryo have been highly conserved during evolution as their vertebrate counterparts, chordin and BMP-4, function homologously to define neural versus non-neural ectoderm in Xenopus. Here we exploit the genetically sensitive adult wing vein pattern to investigate the generality of the antagonistic relationship between sog and dpp. We show that dpp is expressed in vein primordia during pupal wing development and functions to promote vein formation. In contrast, sog is expressed in complementary intervein cells and suppresses vein formation. sog and dpp function during the same phenocritical periods (i.e. 16-28 hours after pupariation) to influence the vein versus intervein cell fate choice. The conflicting activities of dpp and sog are also revealed by antagonistic dosage-sensitive interactions between these two genes during vein development. Analysis of vein and intervein marker expression in dpp and sog mutant wings suggests that dpp promotes vein fates indirectly by activating the vein gene rhomboid (rho), and that sog functions by blocking an autoactivating Dpp feedback loop. These data support the view that Sog is a dedicated Dpp antagonist.

We cloned the dbl-1 gene, a C. elegans homolog of Drosophila decapentaplegic and vertebrate BMP genes. Loss-of-function mutations in dbl-1 cause markedly reduced body size and defective male copulatory structures. Conversely, dbl-1 overexpression causes markedly increased body size and partly complementary male tail phenotypes, indicating that DBL-1 acts as a dose-dependent regulator of these processes. Evidence from genetic interactions indicates that these effects are mediated by a Smad signaling pathway, for which DBL-1 is a previously unidentified ligand. Our study of the dbl-1 expression pattern suggests a role for neuronal cells in global size regulation as well as male tail patterning.

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The Escherichia coli catabolite activator protein (CAP) activates transcription at P(lac), P(gal), and other promoters through interactions with the RNA polymerase alpha subunit carboxyl-terminal domain (alphaCTD). We determined the crystal structure of the CAP-alphaCTD-DNA complex at a resolution of 3.1 angstroms. CAP makes direct protein-protein interactions with alphaCTD, and alphaCTD makes direct protein-DNA interactions with the DNA segment adjacent to the DNA site for CAP. There are no large-scale conformational changes in CAP and alphaCTD, and the interface between CAP and alphaCTD is small. These findings are consistent with the proposal that activation involves a simple "recruitment" mechanism.

1300 independent Ac transposants. The majority of transposed Ac elements are linked to either the bz or the wx donor loci on chromosome 9. A few of the insertions produce obvious visible phenotypes, but most of them do not, suggesting that these populations will be more useful for reverse genetics than for forward transposon mutagenesis. An inverse polymerase chain reaction method was adapted for the isolation of DNA adjacent to the transposed Ac elements (tac sites). Most Ac insertions were into unique DNA. By sequencing tac sites and comparing the sequences to existing databases, insertions were identified in a number of putative maize genes. The expression of most of these genes was confirmed by RNA gel blot analysis. We report here the isolation and characterization of the first 46 tac sites from the two insertion libraries.

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Zea mays L. ssp. mays, or corn, one of the most important crops and a model for plant genetics, has a genome approximately 80% the size of the human genome. To gain global insight into the organization of its genome, we have sequenced the ends of large insert clones, yielding a cumulative length of one-eighth of the genome with a DNA sequence read every 6.2 kb, thereby describing a large percentage of the genes and transposable elements of maize in an unbiased approach. Based on the accumulative 307 Mb of sequence, repeat sequences occupy 58% and genic regions occupy 7.5%. A conservative estimate predicts approximately 59,000 genes, which is higher than in any other organism sequenced so far. Because the sequences are derived from bacterial artificial chromosome clones, which are ordered in overlapping bins, tagged genes are also ordered along continuous chromosomal segments. Based on this positional information, roughly one-third of the genes appear to consist of tandemly arrayed gene families. Although the ancestor of maize arose by tetraploidization, fewer than half of the genes appear to be present in two orthologous copies, indicating that the maize genome has undergone significant gene loss since the duplication event.

BACKGROUND: Members of TGFbeta superfamily are found to play important roles in many cellular processes, such as proliferation, differentiation, development, apoptosis, and cancer. In Drosophila, there are seven ligands that function through combinations of three type I receptors and two type II receptors. These signals can be roughly grouped into two major TGFbeta pathways, the dpp/BMP and activin pathways, which signal primarily through thick veins (tkv) and baboon (babo). Few downstream targets are known for either pathway, especially targets expressed in the Drosophila brain. RESULTS: tkv and babo both affect the growth of tissues, but have varying effects on patterning. We have identified targets for the tkv and babo pathways by employing microarray techniques using activated forms of the receptors expressed in the brain. In these experiments, we compare the similarities of target genes of these two pathways in the brain. About 500 of 13,500 examined genes changed expression at 95% confidence level (P < 0.05). Twenty-seven genes are co-regulated 1.5 fold by both the tkv and babo pathways. These regulated genes cluster into various functional groups such as DNA/RNA binding, signal transducers, enzymes, transcription regulators, and neuronal regulators. RNAi knockdown experiments of homologs of several of these genes show abnormal growth regulation, suggesting these genes may execute the growth properties of TGFbeta. CONCLUSIONS: Our genomic-wide microarray analysis has revealed common targets for the tkv and babo pathways and provided new insights into downstream effectors of two distinct TGFbeta like pathways. Many of these genes are novel and several genes are implicated in growth control. Among the genes regulated by both pathways is ultraspiracle, which further connects TGFbeta with neuronal remodeling.

The unstable mutation bz-m039 arose in a maize (Zea mays) stock that originated from a plant infected with barley stripe mosaic virus. The instability of the mutation is caused by a 3.9-kb mobile element that has been named Jittery (Jit). Jit has terminal inverted repeats (TIRs) of 181 bp, causes a 9-bp direct duplication of the target site, and appears to excise autonomously. It is predicted to encode a single 709-amino acid protein, JITA, which is distantly related to the MURA transposase protein of the Mutator system but is more closely related to the MURA protein of Mutator-like elements (MULEs) from Arabidopsis thaliana and rice (Oryza sativa). Like MULEs, Jit resembles Mutator in the length of the element's TIRs, the size of the target site duplication, and in the makeup of its transposase but differs from the autonomous element Mutator-Don Robertson in that it encodes a single protein. Jit also differs from Mutator elements in the high frequency with which it excises to produce germinal revertants and in its copy number in the maize genome: Jit-like TIRs are present at low copy number in all maize lines and teosinte accessions examined, and JITA sequences occur in only a few maize inbreds. However, Jit cannot be considered a bona fide transposon in its present host line because it does not leave footprints upon excision and does not reinsert in the genome. These unusual mobile element properties are discussed in light of the structure and gene organization of Jit and related elements.

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Maize (Zea mays or corn) plays many varied and important roles in society. It is not only an important experimental model plant, but also a major livestock feed crop and a significant source of industrial products such as sweeteners and ethanol. In this study we report the systematic analysis of contiguous sequences of the maize genome. We selected 100 random regions averaging 144 kb in size, representing about 0.6% of the genome, and generated a high-quality dataset for sequence analysis. This sampling contains 330 annotated genes, 91% of which are supported by expressed sequence tag data from maize and other cereal species. Genes averaged 4 kb in size with five exons, although the largest was over 59 kb with 31 exons. Gene density varied over a wide range from 0.5 to 10.7 genes per 100 kb and genes did not appear to cluster significantly. The total repetitive element content we observed (66%) was slightly higher than previous whole-genome estimates (58%-63%) and consisted almost exclusively of retroelements. The vast majority of genes can be aligned to at least one sequence read derived from gene-enrichment procedures, but only about 30% are fully covered. Our results indicate that much of the increase in genome size of maize relative to rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) is attributable to an increase in number of both repetitive elements and genes.

Methylation on the base or the ribose is prevalent in eukaryotic ribosomal RNAs (rRNAs) and is thought to be crucial for ribosome biogenesis and function. Artificially introduced 2'-O-methyl groups in small interfering RNAs (siRNAs) can stabilize siRNAs in serum without affecting their activities in RNA interference in mammalian cells. Here, we show that plant microRNAs (miRNAs) have a naturally occurring methyl group on the ribose of the last nucleotide. Whereas methylation of rRNAs depends on guide RNAs, the methyltransferase protein HEN1 is sufficient to methylate miRNA/miRNA* duplexes. Our studies uncover a new and crucial step in plant miRNA biogenesis and have profound implications in the function of miRNAs.

MicroRNAs (miRNA) are non-coding small (approximately 22nt) RNAs that regulate diverse physiological and developmental processes. In animals, they regulate target genes by binding imperfectly to 3'UTR sequences in mRNAs and attenuate translation. There are hundreds of miRNA genes in animals, and current studies show they constitute a minimum of 1% of known genes. We are just beginning to understand the diverse roles they play in cellular processes, which include signaling pathways, developmental pathways, and possibly various types of cancers.

MicroRNAs (miRNAs) are post-transcriptional regulators participating in biological processes ranging from differentiation to carcinogenesis. We developed a rational probe design algorithm and a sensitive labelling scheme for optimizing miRNA microarrays. Our microarray contains probes for all validated miRNAs from five species, with the potential for drawing on species conservation to identify novel miRNAs with homologous probes. These methods are useful for high-throughput analysis of micro RNAs from various sources, and allow analysis with limiting quantities of RNA. The system design can also be extended for use on Luminex beads or on 96-well plates in an ELISA-style assay. We optimized hybridization temperatures using sequence variations on 20 of the probes and determined that all probes distinguish wild-type from 2 nt mutations, and most probes distinguish a 1 nt mutation, producing good selectivity between closely-related small RNA sequences. Results of tissue comparisons on our microarrays reveal patterns of hybridization that agree with results from Northern blots and other methods.

Maize (Zea mays or corn), both a major food source and an important cytogenetic model, evolved from a tetraploid that arose about 4.8 million years ago (Mya). As a result, maize has extensive duplicated regions within its genome. We have sequenced the two copies of one such region, generating 7.8 Mb of sequence spanning 17.4 cM of the short arm of chromosome 1 and 6.6 Mb (25.6 cM) from the long arm of chromosome 9. Rice, which did not undergo a similar whole genome duplication event, has only one orthologous region (4.9 Mb) on the short arm of chromosome 3, and can be used as reference for the maize homoeologous regions. Alignment of the three regions allowed identification of syntenic blocks, and indicated that the maize regions have undergone differential contraction in genic and intergenic regions and expansion by the insertion of retrotransposable elements. Approximately 9% of the predicted genes in each duplicated region are completely missing in the rice genome, and almost 20% have moved to other genomic locations. Predicted genes within these regions tend to be larger in maize than in rice, primarily because of the presence of predicted genes in maize with larger introns. Interestingly, the general gene methylation patterns in the maize homoeologous regions do not appear to have changed with contraction or expansion of their chromosomes. In addition, no differences in methylation of single genes and tandemly repeated gene copies have been detected. These results, therefore, provide new insights into the diploidization of polyploid species.

MicroRNAs (miRNAs) are small, abundant transcripts that can bind partially homologous target messages to inhibit their translation in animal cells. miRNAs have been shown to affect a broad spectrum of biological activities, including developmental fate determination, cell signaling and oncogenesis. Little is known, however, of miRNA contributions to aging. We examined the expression of 114 identified Caenorhabditis elegans miRNAs during the adult lifespan and find that 34 miRNAs exhibit changes in expression during adulthood (P<or= 0.05), 31 with more than a twofold level change. The majority of age-regulated miRNAs decline in relative abundance as animals grow older. Expression profiles of developmental timing regulators lin-4 and let-7 miRNAs, as well as conserved muscle miRNA miR-1, show regulation during adulthood. We also used bioinformatic approaches to predict miRNA targets encoded in the C. elegans genome and we highlight candidate miRNA-regulated genes among C. elegans genes previously shown to affect longevity, genes encoding insulin-like ligands, and genes preferentially expressed in C. elegans muscle. Our observations identify miRNAs as potential modulators of age-related decline and suggest a general reduction of message-specific translational inhibition during aging, a previously undescribed feature of C. elegans aging. Since many C. elegans age-regulated miRNAs are conserved across species, our observations identify candidate age-regulating miRNAs in both nematodes and humans.

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Sir2 and Hst1 are NAD(+)-dependent histone deacetylases of budding yeast that are related by strong sequence similarity. Nevertheless, the two proteins promote two mechanistically distinct forms of gene repression. Hst1 interacts with Rfm1 and Sum1 to repress the transcription of specific middle-sporulation genes. Sir2 interacts with Sir3 and Sir4 to silence genes contained within the silent-mating-type loci and telomere chromosomal regions. To identify the determinants of gene-specific versus regional repression, we created a series of Hst1::Sir2 hybrids. Our analysis yielded two dual-specificity chimeras that were able to perform both regional and gene-specific repression. Regional silencing by the chimeras required Sir3 and Sir4, whereas gene-specific repression required Rfm1 and Sum1. Our findings demonstrate that the nonconserved N-terminal region and two amino acids within the enzymatic core domain account for cofactor specificity and proper targeting of these proteins. These results suggest that the differences in the silencing and repression functions of Sir2 and Hst1 may not be due to differences in enzymatic activities of the proteins but rather may be the result of distinct cofactor specificities.

Maize (Zea mays) plants make different types of vegetative or reproductive branches during development. Branches develop from axillary meristems produced on the flanks of the vegetative or inflorescence shoot apical meristem. Among these branches are the spikelets, short grass-specific structures, produced by determinate axillary spikelet-pair and spikelet meristems. We investigated the mechanism of branching in maize by making transgenic plants expressing a native expressed endogenous auxin efflux transporter (ZmPIN1a) fused to yellow fluorescent protein and a synthetic auxin-responsive promoter (DR5rev) driving red fluorescent protein. By imaging these plants, we found that all maize branching events during vegetative and reproductive development appear to be regulated by the creation of auxin response maxima through the activity of polar auxin transporters. We also found that the auxin transporter ZmPIN1a is functional, as it can rescue the polar auxin transport defects of the Arabidopsis (Arabidopsis thaliana) pin1-3 mutant. Based on this and on the groundbreaking analysis in Arabidopsis and other species, we conclude that branching mechanisms are conserved and can, in addition, explain the formation of axillary meristems (spikelet-pair and spikelet meristems) that are unique to grasses. We also found that BARREN STALK1 is required for the creation of auxin response maxima at the flanks of the inflorescence meristem, suggesting a role in the initiation of polar auxin transport for axillary meristem formation. Based on our results, we propose a general model for branching during maize inflorescence development.

The Rice Annotation Project Database (RAP-DB) was created to provide the genome sequence assembly of the International Rice Genome Sequencing Project (IRGSP), manually curated annotation of the sequence, and other genomics information that could be useful for comprehensive understanding of the rice biology. Since the last publication of the RAP-DB, the IRGSP genome has been revised and reassembled. In addition, a large number of rice-expressed sequence tags have been released, and functional genomics resources have been produced worldwide. Thus, we have thoroughly updated our genome annotation by manual curation of all the functional descriptions of rice genes. The latest version of the RAP-DB contains a variety of annotation data as follows: clone positions, structures and functions of 31 439 genes validated by cDNAs, RNA genes detected by massively parallel signature sequencing (MPSS) technology and sequence similarity, flanking sequences of mutant lines, transposable elements, etc. Other annotation data such as Gnomon can be displayed along with those of RAP for comparison. We have also developed a new keyword search system to allow the user to access useful information. The RAP-DB is available at: http://rapdb.dna.affrc.go.jp/ and http://rapdb.lab.nig.ac.jp/.

BACKGROUND: MicroRNAs (miRNAs) are short non-coding RNA molecules that target mRNAs to control gene expression by attenuating the translational efficiency and stability of transcripts. They are found in a wide variety of organisms, from plants to insects and humans. Here, we use Drosophila to investigate the possibility that circadian clocks regulate the expression of miRNAs. RESULTS: We used a microarray platform to survey the daily levels of D. melanogaster miRNAs in adult heads of wildtype flies and the arrhythmic clock mutant cyc01. We find two miRNAs (dme-miR-263a and -263b) that exhibit robust daily changes in abundance in wildtype flies that are abolished in the cyc01 mutant. dme-miR-263a and -263b reach trough levels during the daytime, peak during the night and their levels are constitutively elevated in cyc01 flies. A similar pattern of cycling is also observed in complete darkness, further supporting circadian regulation. In addition, we identified several miRNAs that appear to be constitutively expressed but nevertheless differ in overall daily levels between control and cyc01 flies. CONCLUSION: The circadian clock regulates miRNA expression in Drosophila, although this appears to be highly restricted to a small number of miRNAs. A common mechanism likely underlies daily changes in the levels of dme-miR-263a and -263b. Our results suggest that cycling miRNAs contribute to daily changes in mRNA and/or protein levels in Drosophila. Intriguingly, the mature forms of dme-miR-263a and -263b are very similar in sequence to several miRNAs recently shown to be under circadian regulation in the mouse retina, suggesting conserved functions.