A small region (220 bases) of SV40 sequence information—141 bases before the polyadenylation site and 79 beyond—are sufficient for cleavage of an messenger RNA precursor (that is, the formation of a mature 3′ terminus), the addition of polyadenylic acid, and the transport of messenger RNA from the nucleus to the cytoplasm. These 220 bases include a highly conserved sequence—AAUAAA (A, adenine; U, uracil). Four point mutations in this sequence—AACAAA, AAUUAA, AAUACA, and AAUGAA (C, cytosine; G, guanine)—prevent cleavage.

Structural organization of organs in multicellular organisms occurs through intricate patterning mechanisms that often involve complex interactions between transcription factors in regulatory networks. For example, INDEHISCENT (IND), a basic helix-loop-helix (bHLH) transcription factor, specifies formation of the narrow stripes of valve margin tissue, where Arabidopsis thaliana fruits open on maturity. Another bHLH transcription factor, SPATULA (SPT), is required for reproductive tissue development from carpel margins in the Arabidopsis gynoecium before fertilization. Previous studies have therefore assigned the function of SPT to early gynoecium stages and IND to later fruit stages of reproductive development. Here we report that these two transcription factors interact genetically and via protein-protein contact to mediate both gynoecium development and fruit opening. We show that IND directly and positively regulates the expression of SPT, and that spt mutants have partial defects in valve margin formation. Careful analysis of ind mutant gynoecia revealed slight defects in apical tissue formation, and combining mutations in IND and SPT dramatically enhanced both single-mutant phenotypes. Our data show that SPT and IND at least partially mediate their joint functions in gynoecium and fruit development by controlling auxin distribution and suggest that this occurs through cooperative binding to regulatory sequences in downstream target genes.

Tissue patterning in multicellular organisms is the output of precise spatio-temporal regulation of gene expression coupled with changes in hormone dynamics. In plants, the hormone auxin regulates growth and development at every stage of a plant's life cycle. Auxin signaling occurs through binding of the auxin molecule to a TIR1/AFB F-box ubiquitin ligase, allowing interaction with Aux/IAA transcriptional repressor proteins. These are subsequently ubiquitinated and degraded via the 26S proteasome, leading to derepression of auxin response factors (ARFs). How auxin is able to elicit such a diverse range of developmental responses through a single signaling module has not yet been resolved. Here we present an alternative auxin-sensing mechanism in which the ARF ARF3/ETTIN controls gene expression through interactions with process-specific transcription factors. This noncanonical hormone-sensing mechanism exhibits strong preference for the naturally occurring auxin indole 3-acetic acid (IAA) and is important for coordinating growth and patterning in diverse developmental contexts such as gynoecium morphogenesis, lateral root emergence, ovule development, and primary branch formation. Disrupting this IAA-sensing ability induces morphological aberrations with consequences for plant fitness. Therefore, our findings introduce a novel transcription factor-based mechanism of hormone perception in plants.

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BackgroundThe Brassicaceae family includes the model plant Arabidopsis thaliana as well as a number of agronomically important species such as oilseed crops (in particular Brassica napus, B. juncea and B. rapa) and vegetables (eg. B. rapa and B. oleracea).Separated by only 10-20 million years, Brassica species and Arabidopsis thaliana are closely related, and it is expected that knowledge obtained relating to Arabidopsis growth and development can be translated into Brassicas for crop improvement. Moreover, certain aspects of plant development are sufficiently different between Brassica and Arabidopsis to warrant studies to be carried out directly in the crop species. However, mutating individual genes in the amphidiploid Brassicas such as B. napus and B. juncea may, on the other hand, not give rise to expected phenotypes as the genomes of these species can contain up to six orthologues per single-copy Arabidopsis gene. In order to elucidate and possibly exploit the function of redundant genes for oilseed rape crop improvement, it may therefore be more efficient to study the effects in one of the diploid Brassica species such as B. rapa. Moreover, the ongoing sequencing of the B. rapa genome makes this species a highly attractive model for Brassica research and genetic resource development.ResultsSeeds from the diploid Brassica A genome species, B. rapa were treated with ethyl methane sulfonate (EMS) to produce a TILLING (Targeting Induced Local Lesions In Genomes) population for reverse genetics studies. We used the B. rapa genotype, R-o-18, which has a similar developmental ontogeny to an oilseed rape crop. Hence this resource is expected to be well suited for studying traits with relevance to yield and quality of oilseed rape. DNA was isolated from a total of 9,216 M2 plants and pooled to form the basis of the TILLING platform. Analysis of six genes revealed a high level of mutations with a density of about one per 60 kb. This analysis also demonstrated that screening a 1 kb amplicon in just one third of the population (3072 M2 plants) will provide an average of 68 mutations and a 97% probability of obtaining a stop-codon mutation resulting in a truncated protein. We furthermore calculated that each plant contains on average ~10,000 mutations and due to the large number of plants, it is predicted that mutations in approximately half of the GC base pairs in the genome exist within this population.ConclusionsWe have developed the first EMS TILLING resource in the diploid Brassica species, B. rapa. The mutation density in this population is ~1 per 60 kb, which makes it the most densely mutated diploid organism for which a TILLING population has been published. This resource is publicly available through the RevGenUK reverse genetics platform http://revgenuk.jic.ac.uk.

The sequence AAUAAA is found near the polyadenylation site of eucaryotic mRNAs. This sequence is required for accurate and efficient cleavage and polyadenylation of pre-mRNAs in vivo. In this study we show that synthetic simian virus 40 late pre-mRNAs are cleaved and polyadenylated in vitro in a HeLa cell nuclear extract, and that cleavage in vitro is abolished by each of four different single-base changes in AAUAAA. In this same extract, precleaved RNAs (RNAs with 3' termini at the polyadenylation site) are efficiently polyadenylated. This in vitro polyadenylation reaction also requires the AAUAAA sequence.The 3' termini of eucaryotic mRNAs are formed by endonucleolytic cleavage of an mRNA precursor and the addition of approximately 250 adenylate residues [poly(A)] to the newly formed end (8,22,26,34). The structural features of the precursor that are required for cleavage and polyadenylation have not been completely defined, but are known to include two separable elements, the AAUAAA sequence (10,13,23,27,32) and the downstream element (or TG box) (7,12,19,20,28,29,33).The AAUAAA sequence, located 6 to 26 bases before the poly(A) addition site of mammalian mRNAs, is required for cleavage. Deletions (10) or single-base-pair changes (13,23,32) in AAUAAA prevent cleavage in vivo, but do not prevent polyadenylation; thus mRNA precursors containing mutant AAUAAA sequences are cleaved inefficiently, but all precursors that are cleaved also are polyadenylated (23,32). The downstream element is located 10 to 30 bases beyond the polyadenylation site, in the 3' flanking sequence. mRNA precursors from which this sequence has been deleted are cleaved inefficiently (7,12,19,20,29). The AAUAAA sequence is quantitatively more important than the downstream element. For example, in injected oocytes, cleavage is reduced more than 50-fold by point mutations in AAUAAA (32) but only 5-to 10-fold by deletion of the downstream element (7; D. Zarkower, unpublished data). Similarly, the AAUAAA sequence is more highly conserved than the downstream element (4, 20, 32).Recently Moore and Sharp (25) demonstrated cleavage and polyadenylation of a synthetic adenovirus pre-mRNA in an extract prepared from human cells (9, 25). To establish the physiological relevance of these cell-free reactions, they must be compared with the same reactions in vivo; the in vitro reactions must be accurate and efficient and must depend on the same sequences that are required in vivo. Hart et al. (12) cleavage, polyadenylation is dependent on the AAUAAA sequence.MATERIALS AND METHODS RNA substrates. In vitro transcription was performed as described previously (21), except that the GTP concentration was 300 ,uM and 1.2 mM GpppG (P-L Biochemicals) was included to produce capped transcripts (14). After the transcription reaction, DNA was removed with RNase-free DNase (P-L), and the transcripts were purified by repeated ethanol precipitation in the presence of 2 M ammonium

The interactions of several pyrrolo[2, 1-c][1,4]benzodiazepine (PBD) antitumor antibiotics with linearized plasmid pGEM-2-N-ras DNA have been analyzed by quantitative in vitro transcription (QIVT) and in vitro transcription footprinting (IVTF) methods. A concentration-dependent inhibitory effect of the PBDs on transcription is observed using both techniques. The rank order for overall inhibition of transcription by the QIVT method is found to be: sibiromycin > tomaymycin > anthramycin > DC-81 > neothramycin, whereas the IVTF experiments show a different ranking: sibiromycin > anthramycin > neothramycin > tomaymycin. In addition, stimulation of transcription was observed at low PBD concentrations in both the QIVT and IVTF experiments. These results demonstrate unequivocally that the formation of PBD-DNA adducts at AGA-5' base sequences on the transcribed strand results in transcription blockage for all PBDs examined. Furthermore, the sequence of flanking base pairs appears to influence the degree of blocking, with the sequences ACAGAAA-5', AAAGATG-5', AGAGATA-5', and CAAGAAC-5' providing the most pronounced blocks for all PBDs studied in this system. Neothramycin and tomaymycin cause additional blocks at some GGA-5' and TGA-5' sequences. Parallel MPE-Fe(II) footprinting studies have revealed PBD binding sites on both the transcribing and nontranscribing strands, although all transcription blocks determined from the IVTF assays are due to drug bound on the transcribing DNA template strand.