The structure of the complex of full-length Escherichia coli LexA repressor with a consensus operator DNA fragment has been probed by affinity photo-cross-linking and affinity cleavage. These methods allow the determination of approximate intermolecular distances between a given protein residue and a base or sugar moiety within the operator. In a first step unique cysteine residues were introduced in positions 7, 28, 38, or 52 of the protein. In all four cases, the original amino acid was an arginine. The four amino acids in these positions were expected to be situated on the surface of LexA interacting with DNA, as inferred from the structure of the LexA DNA binding domain [Fogh et al. (1994) EMBO J. 13, 3936-3944]. In a second step, these unique cysteine side chains of the purified proteins were chemically modified either with 4-azidophenacyl bromide or with S-(2-pyridylthio)cysteaminyl-EDTA. The first set of derivatives gives rise to UV-induced cross-linking which may be revealed by alkali/heat treatment; the second leads to direct DNA cleavage in the proximity of the derivatized amino acid. To reduce hydroxyl radical diffusion, the EDTA-iron cleavage reactions were done in the presence of high amounts of glycerol. The results indicate that amino acids 7 and 52 are near nucleotide pairs 8-12 of the operator and that amino acids 28 and 36 of LexA are near nucleotide pairs 5-8 of the operator. The results unambiguously define the orientation of the LexA DNA binding domain relative to the operator and provide support for the model of the LexA-operator complex proposed by Knegtel et al. [(1995) Proteins 21, 226-236]. Ethylation interference experiments further suggest that Arg-7 contacts the phosphate group between nucleotides 8 and 9 as predicted by the model.

We report here the isolation of spectinomycin-resistant mutants in cultured cells of Medicago sativa line RegenSY-T2. Spectinomycin induces bleaching of cultured alfalfa cells due to inhibition of protein synthesis on the prokaryotic type 70S plastid ribosomes. Spontaneous mutants resistant to spectinomycin bleaching were identified by their ability to form green shoots on plant regeneration medium containing selective spectinomycin concentrations in the range of 25-50 mg/l. Sequencing of the plastid rrn16 gene revealed that spectinomycin resistance is due to mutations in a conserved stem structure of the 16S rRNA. Resistant plants transferred to the greenhouse developed normally and produced spectinomycin-resistant seed progeny. In light of their absence in soybean, a related leguminous plant, the isolation of spectinomycin-resistant mutants in M. sativa was unexpected. The new mutations are useful for the study of plastid inheritance, as demonstrated by detection of predominantly paternal plastid inheritance in the RegenSY-T2 x Szapko57 cross, and can be used as selective markers in plastid transformation vectors to obtain cisgenic plants.

150 copies of a transposon-like sequence, termed Heltir, that has terminal inverted repeats resembling Helitron 3' termini. Nonautonomous Helitrons make up at least 2% of the maize genome and most of those tested show +/- polymorphisms among modern inbred lines.

Helitrons represent a new class of transposable elements recently uncovered in plants and animals. One remarkable feature of Helitrons is their ability to capture gene sequences, which makes them of considerable potential evolutionary importance. However, because Helitrons lack the typical structural features of other DNA transposable elements, identifying them is a challenge. Currently, most researchers identify Helitrons manually by comparing sequences. With the maize whole genome sequencing project underway, an automated computational Helitron searching tool is needed. The characterization of Helitron activities in maize needs to be addressed in order to better understand the impact of Helitrons on the organization of the genome.\\ We developed and implemented a heuristic searching algorithm in PERL for identifying Helitrons. Our HelitronFinder program will (i) take FASTA-formatted DNA sequences as input and identify the hairpin looping patterns, and (ii) exploit the consensus 5' and 3' end sequences of known Helitrons to identify putative ends. We randomly selected five predicted Helitrons from the program's high quality output for molecular verification. Four out of the five predicted Helitrons were confirmed by PCR assays and DNA sequencing in different maize inbred lines. The HelitronFinder program identified two head-to-head dissimilar Helitrons in a maize BAC sequence.\\ We have identified 140 new Helitron candidates in maize with our computational tool HelitronFinder by searching maize DNA sequences currently available in GenBank. Four out of five candidates were confirmed to be real by empirical methods, thus validating the predictions of HelitronFinder. Additional points to emerge from our study are that Helitrons do not always insert at an AT dinucleotide in the host sequences, that they can insert immediately adjacent to an existing Helitron, and that their movement may cause changes in the flanking region, such as deletions.

BACKGROUND: Retrotransposons are commonly occurring eukaryotic transposable elements (TEs). Among these, long terminal repeat (LTR) retrotransposons are the most abundant TEs and can comprise 50-90% of the genome in higher plants. By comparing the orthologous chromosomal regions of closely related species, the effects of TEs on the evolution of plant genomes can be studied in detail. RESULTS: Here, we compared the composition and organization of TEs within five orthologous chromosomal regions among three grass species: maize, sorghum, and rice. We identified a total of 132 full or fragmented LTR retrotransposons in these regions. As a percentage of the total cumulative sequence in each species, LTR retrotransposons occupy 45.1% of the maize, 21.1% of the rice, and 3.7% of the sorghum regions. The most common elements in the maize retrotransposon-rich regions are the copia-like retrotransposons with 39% and the gypsy-like retrotransposons with 37%. Using the contiguous sequence of the orthologous regions, we detected 108 retrotransposons with intact target duplication sites and both LTR termini. Here, we show that 74% of these elements inserted into their host genome less than 1 million years ago and that many retroelements expanded in size by the insertion of other sequences. These inserts were predominantly other retroelements, however, several of them were also fragmented genes. Unforeseen was the finding of intact genes embedded within LTR retrotransposons. CONCLUSION: Although the abundance of retroelements between maize and rice is consistent with their different genome sizes of 2,364 and 389 Mb respectively, the content of retrotransposons in sorghum (790 Mb) is surprisingly low. In all three species, retrotransposition is a very recent activity relative to their speciation. While it was known that genes re-insert into non-orthologous positions of plant genomes, they appear to re-insert also within retrotransposons, potentially providing an important role for retrotransposons in the evolution of gene function.

Background: The nonautonomous maize Ds transposons can only move in the presence of the autonomous element Ac. They comprise a heterogeneous group that share 11-bp terminal inverted repeats (TIRs) and some subterminal repeats, but vary greatly in size and composition. Three classes of Ds elements can cause mutations: Ds-del, internal deletions of the 4.6-kb Ac element; Ds1, ~400-bp in size and sharing little homology with Ac, and Ds2, variably-sized elements containing about 0.5 kb from the Ac termini and unrelated internal sequences. Here, we analyze the entire complement of Ds-related sequences in the genome of the inbred B73 and ask whether additional classes of Ds-like (Ds-l) elements, not uncovered genetically, are mobilized by Ac. We also compare the makeup of Ds-related sequences in two maize inbreds of different origin. Results: We found 903 elements with 11-bp Ac/Ds TIRs flanked by 8-bp target site duplications. Three resemble Ac, but carry small rearrangements. The others are much shorter, once extraneous insertions are removed. There are 331 Ds1 and 39 Ds2 elements, many of which are likely mobilized by Ac, and two novel classes of Ds-l elements. Ds-l3 elements lack subterminal homology with Ac, but carry transposase gene fragments, and represent decaying Ac elements. There are 44 such elements in B73. Ds-l4 elements share little similarity with Ac outside of the 11-bp TIR, have a modal length of ~1 kb, and carry filler DNA which, in a few cases, could be matched to gene fragments. Most Ds-related elements in B73 (486/903) fall in this class. None of the Ds-l elements tested responded to Ac. Only half of Ds insertion sites examined are shared between the inbreds B73 and W22. Conclusions: The majority of Ds-related sequences in maize correspond to Ds-l elements that do not transpose in the presence of Ac. Unlike actively transposing elements, many Ds-l elements are inserted in repetitive DNA, where they probably become methylated and begin to decay. The filler DNA present in most elements is occasionally captured from genes, a rare feature in transposons of the hAT superfamily to which Ds belongs. Maize inbreds of different origin are highly polymorphic in their DNA transposon makeup.

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.

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.

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.

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 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.