Signals from transforming growth factor-beta (TGF-beta) ligands are transmitted within the cell by members of the Smad family, which can be grouped into three classes based on sequence similarities. Our previous identification of both class I and II Smads functioning in a single pathway in C. elegans, raised the issue of whether the requirement for Smads derived from different classes is a general feature of TGF-beta signaling. We report here the identification of a new Drosophila class II Smad, Medea, a close homolog of the human tumor-suppressor gene DPC4. Embryos from germline clones of both Medea and Mad (a class I Smad) are ventralized, as are embryos null for the TGF-beta-like ligand decapentaplegic (dpp). Loss of Medea also blocks dpp signaling during later development, suggesting that Medea, like Mad, is universally required for dpp signaling. Furthermore, we show that the necessity for these two closely related, non-redundant Smads, is due to their different signaling properties - upon activation of the Dpp pathway, Mad is required to actively translocate Medea into the nucleus. These results provide a paradigm for, and distinguish between, the requirement for class I and II Smads in Dpp/BMP signaling.

Two instances of genetic transmission of spontaneous epimutation of the maize P-rr gene were identified. Transmission gave rise to two similar, moderately stable alleles, designated P-pr-1 and P-pr-2, that exhibited Mendelian behavior. Both isolates of P-pr conditioned a variable and variegated phenotype, unlike the uniform pigmentation conditioned by P-rr. Extensive genomic analysis failed to reveal insertions, deletions or restriction site polymorphisms between the new allele and its progenitor. However, methylation of the P gene was increased in P-pr relative to P-rr, and was greatly reduced (though not lost) in a revertant to uniform pigmentation. Variability in pigmentation conditioned by P-pr correlated with variability in transcript levels of the P gene, and both correlated inversely with variability in its methylation. Part of the variability in methylation could be accounted for by a developmental decrease in methylation in all tissues of plants carrying P-pr. We hypothesize that the variegated phenotype results from a general epigenetic pathway which causes a progressive decrease in methylation and increase in expression potential of the P gene as a function of cell divisions in each meristem of the plant. This renders all tissues chimeric for a functional gene; chimerism is visualized as variegation only in pericarp due to the tissue specificity of P gene expression. Therefore, this allele that originates from epimutation may exemplify an epigenetic mechanism for variegation in maize.

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The light-driven steps in the biogenesis and repair of the inorganic core comprising the O-2-evolving center of oxygenic photosynthesis (photosystem II water-oxidation complex, PSII-WOC) are reviewed. These steps, known collectively as photoactivation, involve the photoassembly of the free inorganic cofactors to the cofactor-depleted PSII-(apo-WOC) driven by light and produce the active O-2-evolving core comprised of Mn4CaOxCly. We focus on the functional role of the inorganic components as seen through the competition with non-native cofactors ("inorganic mutants") on water oxidation activity, the rate of the photoassembly reaction, and on structural insights gained from EPR spectroscopy of trapped intermediates formed in the initial steps of the assembly reaction. A chemical mechanism for the initial steps in photoactivation is given that is based on these data. Photoactivation experiments offer the powerful insights gained from replacement of the native cofactors, which together with the recent X-ray structural data for the resting holoenzyme provide a deeper understanding of the chemistry of water oxidation. We also review some new directions in research that photoactivation studies have inspired that look at the evolutionary history of this remarkable catalyst. (c) 2007 Elsevier B.V. All rights reserved.

The multisubunit RNA polymerase (RNAP) in bacteria consists of a catalytically active core enzyme (alpha(2)beta beta'omega) complexed with a sigma factor that is required for promoter-specific transcription initiation. During early elongation the stability of interactions between sigma and core decreases, in part because of the nascent RNA-mediated destabilization of an interaction between region 4 of sigma and the flap domain of the beta-subunit (beta-flap). The nascent RNA-mediated destabilization of the sigma region 4/beta-flap interaction is required for the bacteriophage lambda Q antiterminator protein (lambdaQ) to engage the RNAP holoenzyme. Here, we provide an explanation for this requirement by showing that lambdaQ establishes direct contact with the beta-flap during the engagement process, thus competing with sigma(70) region 4 for access to the beta-flap. We also show that lambdaQ's affinity for the beta-flap is calibrated to ensure that lambdaQ activity is restricted to the lambda late promoter P(R'). Specifically, we find that strengthening the lambdaQ/beta-flap interaction allows lambdaQ to bypass the requirement for specific cis-acting sequence elements, a lambdaQ-DNA binding site and a RNAP pause-inducing element, that normally ensure lambdaQ is recruited exclusively to transcription complexes associated with P(R'). Our findings demonstrate that the beta-flap can serve as a direct target for regulators of elongation.

The bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytic core enzyme (alpha(2)betabeta'omega) in complex with a sigma factor that is essential for promoter recognition and transcription initiation. During early elongation, the stability of interactions between sigma and the remainder of the transcription complex decreases. Nevertheless, there is no mechanistic requirement for release of sigma upon the transition to elongation. Furthermore, sigma can remain associated with RNAP during transcription elongation and influence regulatory events that occur during transcription elongation. Here we demonstrate that promoter-like DNA sequence elements within the initial transcribed region that are known to induce early elongation pausing through sequence-specific interactions with sigma also function to increase the sigma content of downstream elongation complexes. Our findings establish sigma-dependent pausing as a mechanism by which initial transcribed region sequences can influence the composition and functional properties of the transcription elongation complex over distances of at least 700 base pairs.

The bacterial RNA polymeras holoenzyme consists of a catalytic core enzyme in complex with a sigma factor that is required for promoter-specific transcription initiation. During initiation, members of the sigma(70) family of sigma factors contact two conserved promoter elements, the -10 and -35 elements, which are separated by approximately 17 base pairs (bp). sigma(70) family members contain four flexibly linked domains. Two of these domains, sigma(2) and sigma(4), contain determinants for interactions with the promoter -10 and -35 elements respectively. sigma(2) and sigma(4) also contain core-binding determinants. When bound to core the inter-domain distance between sigma(2) and sigma(4) matches the distance between promoter elements separated by approximately 17 bp. Prior work indicates that during early elongation the nascent RNA-assisted displacement of sigma(4) from core can enable the holoenzyme to adopt a configuration in which sigma(2) and sigma(4) are bound to 'promoter-like' DNA elements separated by a single base pair. Here we demonstrate that holoenzyme can also adopt configurations in which sigma(2) and sigma(4) are bound to 'promoter-like' DNA elements separated by 0, 2 or 3 bp. Thus, our findings suggest that displacement of sigma(4) from core enables the RNA polymerase holoenzyme to adopt a broad range of 'elongation-specific' configurations.

To mitigate some of the potentially deleterious environmental and agricultural consequences associated with current land-based-biofuel feedstocks, we propose the use of biofuels derived from aquatic microbial oxygenic photoautotrophs (AMOPs), more commonly known as cyanobacteria, algae, and diatoms. Herein we review their demonstrated productivity in mass culturing and aspects of their physiology that are particularly attractive for integration into renewable biofuel applications. Compared with terrestrial crops, AMOPs are inherently more efficient solar collectors, use less or no land, can be converted to liquid fuels using simpler technologies than cellulose, and offer secondary uses that fossil fuels do not provide. AMOPs pose a new set of technological challenges if they are to contribute as biofuel feedstocks.

The evolution of O-2-producing cyanobacteria that use water as terminal reductant transformed Earth's atmosphere to one suitable for the evolution of aerobic metabolism and complex life. The innovation of water oxidation freed photosynthesis to invade new environments and visibly changed the face of the Earth. We offer a new hypothesis for how this process evolved, which identifies two critical roles for carbon dioxide in the Archean period. First, we present a thermodynamic analysis showing that bicarbonate (formed by dissolution of CO2) is a more efficient alternative substrate than water for O-2 production by oxygenic phototrophs. This analysis clarifies the origin of the long debated "bicarbonate effect" on photosynthetic O-2 production. We propose that bicarbonate was the thermodynamically preferred reductant before water in the evolution of oxygenic photosynthesis. Second, we have examined the speciation of manganese(II) and bicarbonate in water, and find that they form Mn-bicarbonate clusters as the major species under conditions that model the chemistry of the Archean sea. These clusters have been found to be highly efficient precursors for the assembly of the tetramanganese-oxide core of the water-oxidizing enzyme during biogenesis. We show that these clusters can be oxidized at electrochemical potentials that are accessible to anoxygenic phototrophs and thus the most likely building blocks for assembly of the first O-2 evolving photoreaction center, most likely originating from green nonsulfur bacteria before the evolution of cyanobacteria.

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The Xanthomonas campestris catabolite gene activator protein-like protein (CLP) can substitute for the Escherichia coli catabolite gene activator protein (CAP) in transcription activation at the lac promoter (V. de Crecy-Lagard, P. Glaser, P. Lejeune, O. Sismeiro, C. Barber, M. Daniels, and A. Danchin, J. Bacteriol. 172:5877-5883, 1990). We show that CLP has the same DNA binding specificity as CAP at positions 5, 6, and 7 of the DNA half site. In addition, we show that the amino acids at positions 1 and 2 of the recognition helix of CLP are identical to the amino acids at positions 1 and 2 of the recognition helix of CAP:i.e., Arg at position 1 and Glu at position 2.

Myc binds to a 6 bp 2-fold symmetric DNA site: 5'-C-3A-2C-1G+1T+2G+3-3'. Using site-specific 5-bromouracil mediated photocrosslinking, we show that His336 of Myc contacts, or is close to, the thymine 5-methyl group at 2-fold symmetry-related positions -2 and +2 of the DNA site in the Myc-DNA complex. Our results strongly suggest that homologous amino acids of Myc and Max make equivalent contacts in the respective protein-DNA complexes.

Although maize (Zea mays) retrotransposons are recombinationally inert, the highly polymorphic structure of maize haplotypes raises questions regarding the local effect of intergenic retrotransposons on recombination. To examine this effect, we compared recombination in the same genetic interval with and without a large retrotransposon cluster. We used three different bz1 locus haplotypes, McC, B73, and W22, in the same genetic background. We analyzed recombination between the bz1 and stc1 markers in heterozygotes that differ by the presence and absence of a 26-kb intergenic retrotransposon cluster. To facilitate the genetic screen, we used Ds and Ac markers that allowed us to identify recombinants by their seed pigmentation. We sequenced 239 recombination junctions and assigned them to a single nucleotide polymorphism-delimited interval in the region. The genetic distance between the markers was twofold smaller in the presence of the retrotransposon cluster. The reduction was seen in bz1 and stc1, but no recombination occurred in the highly polymorphic intergenic region of either heterozygote. Recombination within genes shuffled flanking retrotransposon clusters, creating new chimeric haplotypes and either contracting or expanding the physical distance between markers. Our findings imply that haplotype structure will profoundly affect the correlation between genetic and physical distance for the same interval in maize.

Recombinants isolated from most meiotic intragenic recombination experiments in maize, but not in yeast, are borne principally on crossover chromosomes. This excess of crossovers is not explained readily by the canonical double-strand break repair model of recombination, proposed to account for a large body of yeast data, which predicts that crossovers (COs) and noncrossovers (NCOs) should be recovered equally. An attempt has been made here to identify general rules governing the recovery of the CO and NCO classes of intragenic recombinants in maize. Recombination was analyzed in bz heterozygotes between a variety of mutations derived from the same or different progenitor alleles. The mutations include point mutations, transposon insertions, and transposon excision footprints. Consequently, the differences between the bz heteroalleles ranged from just two nucleotides to many nucleotides, indels, and insertions. In this article, allelic pairs differing at only two positions are referred to as dimorphic to distinguish them from polymorphic pairs, which differ at multiple positions. The present study has revealed the following effects at these bz heteroalleles: (1) recombination between polymorphic heteroalleles produces mostly CO chromosomes; (2) recombination between dimorphic heteroalleles produces both CO and NCO chromosomes, in ratios apparently dependent on the nature of the heteroalleles; and (3) in dimorphic heterozygotes, the two NCO classes are recovered in approximately equal numbers when the two mutations are point mutations but not when one or both mutations are insertions. These observations are discussed in light of a recent version of the double-strand break repair model of recombination that postulates separate pathways for the formation of CO and NCO products.

Recent genome sequencing efforts have revealed how extensively transposable elements (TEs) have contributed to the shaping of present day plant genomes. DNA transposons associate preferentially with the euchromatic or genic component of plant genomes and have had the opportunity to interact intimately with the genes of the plant host. These interactions have resulted in TEs acquiring host sequences, forming chimeric genes through exon shuffling, replacing regulatory sequences, mobilizing genes around the genome, and contributing genes to the host. The close interaction of transposons with genes has also led to the evolution of intricate cellular mechanisms for silencing transposon activity. Transposons have thus become important subjects of study in understanding epigenetic regulation and, in cases where transposons have amplified to high numbers, how to escape that regulation.