Palaeogenomics of plants: synteny-based modelling of extinct ancestors.
Trends Plant Sci. 15:479-87. Abstract
In the past ten years, international initiatives have led to the development of large sets of genomic resources that allow comparative genomic studies between plant genomes at a high level of resolution. Comparison of map-based genomic sequences revealed shared intra-genomic duplications, providing new insights into the evolution of flowering plant genomes from common ancestors. Plant genomes can be presented as concentric circles, providing a new reference for plant chromosome evolutionary relationships and an efficient tool for gene annotation and cross-genome markers development. Recent palaeogenomic data demonstrate that whole-genome duplications have provided a motor for the evolutionary success of flowering plants over the last 50-70 million years.
Paramutagenicity of a p1 epiallele in maize.
Theoretical and Applied Genetics. (Epub Sep 18) Abstract
Complex silencing mechanisms in plants and other kingdoms target transposons, repeat sequences, invasive viral nucleic acids and transgenes, but also endogenous genes and genes involved in paramutation. Paramutation occurs in a heterozygote when a transcriptionally active allele heritably adopts the epigenetic state of a transcriptionally and/or post-transcriptionally repressed allele. P1-rr and its silenced epiallele P1-pr, which encode a Myb-like transcription factor mediating pigmentation in floral organs of Zea mays, differ in their cytosine methylation pattern and chromatin structure at a complex enhancer site. Here, we tested whether P1-pr is able to heritably silence its transcriptionally active P1-rr allele in a heterozygote and whether DNA methylation is associated with the establishment and maintenance of P1-rr silencing. We found that P1-pr participates in paramutation as the repressing allele and P1-rr as the sensitive allele. Silencing of P1-rr is highly variable compared to the inducing P1-pr resulting in a wide range of gene expression. Whereas cytosine methylation at P1-rr is negatively correlated with transcription and pigment levels after segregation of P1-pr, methylation lags behind the establishment of the repressed p1 gene expression. We propose a model in which P1-pr paramutation is triggered by changing epigenetic states of transposons immediately adjacent to a P1-rr enhancer sequence. Considering the vast amount of transposable elements in the maize genome close to regulatory elements of genes, numerous loci could undergo paramutation-induced allele silencing, which could also have a significant impact on breeding agronomically important traits.
A Pathway for Synapsis Initiation during Zygotene in Drosophila Oocytes.
Curr Biol. 21:1852-7. Abstract
Formation of the synaptonemal complex (SC), or synapsis, between homologs in meiosis is essential for crossing over and chromosome segregation [1-4]. How SC assembly initiates is poorly understood but may have a critical role in ensuring synapsis between homologs and regulating double-strand break (DSB) and crossover formation. We investigated the genetic requirements for synapsis in Drosophila and found that there are three temporally and genetically distinct stages of synapsis initiation. In "early zygotene" oocytes, synapsis is only observed at the centromeres. We also found that nonhomologous centromeres are clustered during this process. In "mid-zygotene" oocytes, SC initiates at several euchromatic sites. The centromeric and first euchromatic SC initiation sites depend on the cohesion protein ORD. In "late zygotene" oocytes, SC initiates at many more sites that depend on the Kleisin-like protein C(2)M. Surprisingly, late zygotene synapsis initiation events are independent of the earlier mid-zygotene events, whereas both mid and late synapsis initiation events depend on the cohesin subunits SMC1 and SMC3. We propose that the enrichment of cohesion proteins at specific sites promotes homolog interactions and the initiation of euchromatic SC assembly independent of DSBs. Furthermore, the early euchromatic SC initiation events at mid-zygotene may be required for DSBs to be repaired as crossovers.
Pattern of diversity in the genomic region near the maize domestication gene tb1.
Proceedings of the National Academy of Sciences of the United States of America. 101:700-7. Abstract
Domesticated maize and its wild ancestor (teosinte) differ strikingly in morphology and afford an opportunity to examine the connection between strong selection and diversity in a major crop species. The tb1 gene largely controls the increase in apical dominance in maize relative to teosinte, and a region of the tb1 locus 5' to the transcript sequence was a target of selection during maize domestication. To better characterize the impact of selection at a major "domestication" locus, we have sequenced the upstream tb1 genomic region and systematically sampled nucleotide diversity for sites located as far as 163 kb upstream to tb1. Our analyses define a selective sweep of approximately 60-90 kb 5' to the tb1 transcribed sequence. The selected region harbors a mixture of unique sequences and large repetitive elements, but it contains no predicted genes. Diversity at the nearest 5' gene to tb1 is typical of that for neutral maize loci, indicating that selection at tb1 has had a minimal impact on the surrounding chromosomal region. Our data also show low intergenic linkage disequilibrium in the region and suggest that selection has had a minor role in shaping the pattern of linkage disequilibrium that is observed. Finally, our data raise the possibility that maize-like tb1 haplotypes are present in extant teosinte populations, and our findings also suggest a model of tb1 gene regulation that differs from traditional views of how plant gene expression is controlled.
Phenyl-azide-mediated photocrosslinking analysis of Cro-DNA interaction..
Journal of molecular biology. 230(2):453-60. Abstract
Using phenyl-azide-mediated photocrosslinking, we show that the alpha carbon of amino acid 2 of the helix-turn-helix motif of bacteriophage lambda Cro is within 12 A of the bottom-strand nucleotides at positions 2 and 3 of the DNA half site in the Cro-DNA complex in solution. This result is in excellent agreement with the crystallographic structure of the Cro-DNA complex. The results of phenyl-azide-mediated photocrosslinking analysis of Cro-DNA interaction, together with the previously reported results of phenyl-azide-mediated photocrosslinking analysis of CAP-DNA interaction, establish that phenyl-azide-mediated photocrosslinking is generalizable and provide information regarding the structural requirements for phenyl-azide-mediated photocrosslinking. Comparison of the results of phenyl-azide-mediated photocrosslinking to the results of EDTA: iron-mediated affinity cleaving indicates that phenyl-azide-mediated photocrosslinking yields superior resolution.
Phosphorylation-independent repression of Yorkie in Fat-Hippo signaling.
Developmental biology. 335:188-97. Abstract
The Fat-Hippo signaling pathway plays an important role in the regulation of normal organ growth during development, and in pathological growth during cancer. Fat-Hippo signaling controls growth through a transcriptional co-activator protein, Yorkie. A Fat-Hippo pathway has been described in which Yorkie is repressed by phosphorylation, mediated directly by the kinase Warts and indirectly by upstream tumor suppressors that promote Warts kinase activity. We present here evidence for an alternate pathway in which Yorkie activity is repressed by direct physical association with three other pathway components: Expanded, Hippo, and Warts. Each of these Yorkie repressors contains one or more PPXY sequence motifs, and associates with Yorkie via binding of these PPXY motifs to WW domains of Yorkie. This direct binding inhibits Yorkie activity independently from effects on Yorkie phosphorylation, and does so both in vivo and in cultured cell assays. These results emphasize the importance of the relative levels of Yorkie and its upstream tumor suppressors to Yorkie regulation, and suggest a dual repression model, in which upstream tumor suppressors can regulate Yorkie activity both by promoting Yorkie phosphorylation and by direct binding.
Photoassembly of the water-oxidizing complex in photosystem II.
Coordination Chemistry Reviews. 252:347-360. Abstract
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.
Photosynthesis: a blueprint for solar energy capture and biohydrogen production technologies.
Photochemical & Photobiological Sciences. 4:957-970. Abstract
Solar energy capture, conversion into chemical energy and biopolymers by photoautotrophic organisms, is the basis for almost all life on Earth. A broad range of organisms have developed complex molecular machinery for the efficient conversion of sunlight to chemical energy over the past 3 billion years, which to the present day has not been matched by any man-made technologies. Chlorophyll photochemistry within photosystem II (PSII) drives the water-splitting reaction efficiently at room temperature, in contrast with the thermal dissociation reaction that requires a temperature of ca. 1550 K. The successful elucidation of the high-resolution structure of PSII, and in particular the structure of its Mn4Ca cluster (K. N. Ferreira, T. M. Iverson, K. Maghlaoui, J. Barber and S. Iwata, Science, 2004, 303, 1831-1838, ref. 1) provides an invaluable blueprint for designing solar powered biotechnologies for the future. This knowledge, combined with new molecular genetic tools, fully sequenced genomes, and an ever increasing knowledge base of physiological processes of oxygenic phototrophs has inspired scientists from many countries to develop new biotechnological strategies to produce renewable CO2-neutral energy from sunlight. This review focuses particularly on the potential of use of cyanobacteria and microalgae for biohydrogen production. Specifically this article reviews the predicted size of the global energy market and the constraints of global warming upon it, before detailing the complex set of biochemical pathways that underlie the photosynthetic process and how they could be modified for improved biohydrogen production.
Physical and Genetic Structure of the Maize Genome Reflects Its Complex Evolutionary History.
PLoS Genet. 3:e123. Abstract
Maize (Zea mays L.) is one of the most important cereal crops and a model for the study of genetics, evolution, and domestication. To better understand maize genome organization and to build a framework for genome sequencing, we constructed a sequence-ready fingerprinted contig-based physical map that covers 93.5% of the genome, of which 86.1% is aligned to the genetic map. The fingerprinted contig map contains 25,908 genic markers that enabled us to align nearly 73% of the anchored maize genome to the rice genome. The distribution pattern of expressed sequence tags correlates to that of recombination. In collinear regions, 1 kb in rice corresponds to an average of 3.2 kb in maize, yet maize has a 6-fold genome size expansion. This can be explained by the fact that most rice regions correspond to two regions in maize as a result of its recent polyploid origin. Inversions account for the majority of chromosome structural variations during subsequent maize diploidization. We also find clear evidence of ancient genome duplication predating the divergence of the progenitors of maize and rice. Reconstructing the paleoethnobotany of the maize genome indicates that the progenitors of modern maize contained ten chromosomes.
Pioneer axon guidance by UNC-129, a C. elegans TGF-β.
Science (New York, NY). 281:706-9. Abstract
The unc-129 gene, like the unc-6 netrin gene, is required to guide pioneer motoraxons along the dorsoventral axis of Caenorhabditis elegans. unc-129 encodes a member of the transforming growth factor-beta (TGF-beta) superfamily of secreted signaling molecules and is expressed in dorsal, but not ventral, rows of body wall muscles. Ectopic expression of UNC-129 from ventral body wall muscle disrupts growth cone and cell migrations that normally occur along the dorsoventral axis. Thus, UNC-129 mediates expression of dorsoventral polarity information required for axon guidance and guided cell migrations in C. elegans.
Plastid genomes in a regenerating tobacco shoot derive from a small number of copies selected through a stochastic process.
Plant J.. 56:975-83. Abstract
The plastid genome (ptDNA) of higher plants is highly polyploid, and the 1000-10 000 copies are compartmentalized with up to approximately 100 plastids per cell. The problem we address here is whether or not a newly arising genome can be established in a developing tobacco shoot, and be transmitted to the seed progeny. We tested this by generating two unequal ptDNA populations in a cultured tobacco cell. The parental tobacco plants in this study have an aurea (yellowish-golden) leaf color caused by the presence of a bar(au) gene in the ptDNA. In addition, the ptDNA carries an aadA gene flanked with the phiC31 phage site-specific recombinase (Int) attP/attB target sites. The genetically distinct ptDNA copies were obtained by Int, which either excised only the aadA marker gene (i.e. did not affect the aurea phenotype) or triggered the deletion of both the aadA and bar(au) transgenes, and thereby restored the green color. The ptDNA determining green plastids represented only a small fraction of the population and was not seen in a transient excision assay, and yet three out of the 53 regenerated shoots carried green plastids in all developmental layers. The remaining 49 Int-expressing plants had either exclusively aurea (24) or variegated (25) leaves with aurea and green sectors. The formation of homoplastomic green shoots with the minor green ptDNA in all developmental layers suggests that the ptDNA population in a regenerating shoot apical meristem derives from a small number of copies selected through a stochastic process.
Plastid marker gene excision by the phiC31 phage site-specific recombinase.
Plant Mol. Biol.. 64:137-43. Abstract
Marker genes are essential for selective amplification of rare transformed plastid genome copies to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here we report excision of plastid marker genes by the phiC31 phage site-specific integrase (Int) that mediates recombination between bacterial (attB) and phage (attP) attachment sites. We tested marker gene excision in a two-step process. First we transformed the tobacco plastid genome with the pCK2 vector in which the spectinomycin resistance (aadA) marker gene is flanked with suitably oriented attB and attP sites. The transformed plastid genomes were stable in the absence of Int. We then transformed the nucleus with a gene encoding a plastid-targeted Int that led to efficient marker gene excision. The aadA marker free Nt-pCK2-Int plants were resistant to phosphinothricin herbicides since the pCK2 plastid vector also carried a bar herbicide resistance gene that, due to the choice of its promoter, causes a yellowish-golden (aurea) phenotype. Int-mediated marker excision reported here is an alternative to the currently used CRE/loxP plastid marker excision system and expands the repertoire of the tools available for the manipulation of the plastid genome.
Plastid transformation in flowering plants.
Genomics of Chloroplasts and Mitochondria. 35:393-414. Abstract
The plastid genome of higher plants is relatively small, 120–230-kb in size, and present in up to 10,000 copies per cell. Standard protocols for the introduction of transforming DNA employ biolistic DNA delivery or polyethylene glycol treatment. Genetically stable, transgenic plants are obtained by modification of the plastid genome by homologous recombination, followed by selection for the transformed genome copy by the expression of marker genes that protect the cells from selective agents. Commonly used selective agents are antibiotics, including spectinomycin, streptomycin, kanamycin and chloramphenicol. Selection for resistance to amino acid analogues has also been successful. The types of plastid genome manipulations include gene deletion, gene insertion, and gene replacement, facilitated by specially designed transformation vectors. Methods are also available for post-transformation removal of marker genes. The model species for plastid genetic manipulation is Nicotiana tabacum, in which most protocols have been tested. Plastid transformation is also available in several solanaceous crops (tomato, potato, eggplant) and ornamental species (petunia, Nicotianasylvestris). Significant progress has been made with Brasssicaceae including cabbage, oilseed rape and Arabidopsis. Recent additions to the crops in which plastid transformation is reproducibly obtained are lettuce, soybean and sugar beet. The monocots are a taxonomic group recalcitrant to plastid transformation; initial inroads have been made only in rice.
The polychromatic Helitron landscape of the maize genome.
Proc. Natl. Acad. Sci. U.S.A.. 106:19916–19921. Abstract
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.
Post-transcriptional regulation of methionine content in maize kernels.
Molecular & general genetics : MGG. 225:331-9. Abstract
Message levels for a methionine-rich 10 kDa zein were determined in three inbred lines of maize and their reciprocal crosses at various stages during endosperm development. Inbred line BSSS-53, which overexpresses the 10 kDa protein in mature kernels, was shown to have higher mRNA levels in developing endosperm, as compared to inbred lines W23 and W64A. Differences in mRNA levels could not be explained by differences in transcription rate of the 10 kDa zein gene, indicating differential post-transcriptional regulation of this storage protein in the different inbred lines analyzed. Among progeny segregating for the BSSS-53 allele of the 10 kDa zein structural gene Zps10/(22), mRNA levels are independent of Zps10/(22) segregation, indicating that post-transcriptional regulation of mRNA levels takes place via a trans-acting mechanism. In the same progeny, mRNA levels are also independent of allelic segregation of the regulatory locus Zpr10/(22). Thus, the trans-acting factor encoded by Zpr10/(22) determines accumulation of 10 kDa zein at a translational or post-translational step. Multiple trans-acting factors are therefore involved in post-transcriptional regulation of the methionine-rich 10 kDa zein.
Primary structure of a genomic zein sequence of maize.
The EMBO journal. 1:1337-42. Abstract
The nucleotide sequence of a genomic clone (termed Z4 ) of the zein multigene family was compared to the nucleotide sequence of related cDNA clones of zein mRNAs. A tandem duplication of a 96-bp sequence is found in the genomic clone that is not present in the related cDNA clones. When the duplication is disregarded, the nucleotide sequence homology between Z4 and its related cDNAs was approximately 97%. The nucleotide sequence is also compared to other isolated cDNAs. No introns in the coding region of the zein gene are detected. The first nucleotide of a putative TATA box, TATAAATA , was located 88 nucleotides upstream of the first nucleotide of the first ATG codon which initiated the open reading frame. The first nucleotide of a putative CCAAT box, CAAAAT , appeared 45 nucleotides upstream of the first nucleotide of the zein cDNA clones in the 3' non-coding region also appeared in the genomic sequence at the same locations. The amino acid composition of the polypeptide specified by the Z4 nucleotide sequence is similar to the known composition of zein proteins.
The primary structure of a plant storage protein: zein.
Nucleic acids research. 9:5163-74. Abstract
The protein sequence of a representative of the zeins, the major storage proteins of maize, has been derived from the nucleotide sequence of a zein cDNA clone. This cDNA was sequence both by the Maxam and Gilbert and the M13-dideoxy techniques. The nucleotide sequence encompasses the non-translated 3' terminus of the mRNA, the entire coding sequence specifying both the mature zein protein and a small signal peptide, and a portion of the non-translated 5' region. The deduced amino acid composition and the amino-terminal amino acid sequence closely resemble those derived from chemical analysis of the zein protein fraction. The data presented represent the first complete amino acid sequence of a plant storage protein.
Processing and phosphorylation of the Fat receptor.
Proceedings of the National Academy of Sciences of the United States of America. Abstract
The Drosophila tumor suppressors fat and discs overgrown (dco) function within an intercellular signaling pathway that controls growth and polarity. fat encodes a transmembrane receptor, but post-translational regulation of Fat has not been described. We show here that Fat is subject to a constitutive proteolytic processing, such that most or all cell surface Fat comprises a heterodimer of stably associated N- and C-terminal fragments. The cytoplasmic domain of Fat is phosphorylated, and this phosphorylation is promoted by the Fat ligand Dachsous. dco encodes a kinase that influences Fat signaling, and Dco is able to promote the phosphorylation of the Fat intracellular domain in cultured cells and in vivo. Evaluation of dco mutants indicates that they affect Fat's influence on growth and gene expression but not its influence on planar cell polarity. Our observations identify processing and phosphorylation as post-translational modifications of Fat, correlate the phosphorylation of Fat with its activation by Dachsous in the Fat-Warts pathway, and enhance our understanding of the requirement for Dco in Fat signaling.