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Luca Comai

Publications and source records attributed to Luca Comai.

At least 19 recordsLinked to original sources
PubMed2006

Molecular karyotyping and aneuploidy detection in Arabidopsis thaliana using quantitative fluorescent polymerase chain reaction.

Certain cellular processes are sensitive to changes in gene dosage. Aneuploidy is deleterious because of an imbalance of gene dosage on a chromosomal scale. Identification, classification and characterization of aneuploidy are therefore important for molecular, population and medical genetics and for a deeper understanding of the mechanisms underlying dosage sensitivity. Notwithstanding recent progress in genomic technologies, limited means are available for detecting and classifying changes in chromosome dose. The development of an inexpensive and scalable karyotyping method would allow rapid detection and characterization of both simple and complex aneuploid types. In addition to the problem of karyotyping, genomic and molecular genetic studies of aneuploids and polyploids are complicated by multiple heterozygous combinations possible at loci present in more than two copies. Quantitative scoring of allele genotypes would enable large-scale population genetic experiments in polyploids, and permit genetic analyses on bulked populations in diploid species. Here, we demonstrate that quantitative fluorescent-polymerase chain reaction (QF-PCR) can be used to simultaneously genotype and karyotype aneuploid and polyploid Arabidopsis thaliana. Comparison of QF-PCR with flow cytometric determination of nuclear DNA content indicated near perfect agreement between the methods, but complete karyotype resolution was only possible using QF-PCR. A complex karyotype, determined by QF-PCR, was validated by comparative genomic hybridization to microarrays. Finally, we screened the progeny of tetraploid individuals and found that more than 25% were aneuploid and that our artificially induced tetraploid strain produced fewer aneuploid individuals than a tetraploid strain isolated from nature.

Alleles↗
PubMed2006

High-throughput discovery of rare human nucleotide polymorphisms by Ecotilling.

Human individuals differ from one another at only approximately 0.1% of nucleotide positions, but these single nucleotide differences account for most heritable phenotypic variation. Large-scale efforts to discover and genotype human variation have been limited to common polymorphisms. However, these efforts overlook rare nucleotide changes that may contribute to phenotypic diversity and genetic disorders, including cancer. Thus, there is an increasing need for high-throughput methods to robustly detect rare nucleotide differences. Toward this end, we have adapted the mismatch discovery method known as Ecotilling for the discovery of human single nucleotide polymorphisms. To increase throughput and reduce costs, we developed a universal primer strategy and implemented algorithms for automated band detection. Ecotilling was validated by screening 90 human DNA samples for nucleotide changes in 5 gene targets and by comparing results to public resequencing data. To increase throughput for discovery of rare alleles, we pooled samples 8-fold and found Ecotilling to be efficient relative to resequencing, with a false negative rate of 5% and a false discovery rate of 4%. We identified 28 new rare alleles, including some that are predicted to damage protein function. The detection of rare damaging mutations has implications for models of human disease.

Algorithms↗
PubMed2006

Parent-dependent loss of gene silencing during interspecies hybridization.

Speciation depends on the establishment of reproductive barriers that allow populations to diverge from each other. Such divergence may involve protein sequence, copy number, or expression changes that are predicted to result in dosage-dependent effects. In plants, such as Arabidopsis thaliana and A. arenosa, postzygotic species barriers often affect seed abortion, and hybrid failure resembles that of interploidy crosses where the paternal genome is in excess. We used this species pair to explore the relationship between hybrid incompatibility and gene silencing. In incompatible crosses, the normally silenced and heterochromatic element ATHILA was expressed from the paternal, but not maternal, chromosomes. Three Polycomb-regulated genes; PHERES1, MEIDOS, and MEDEA, were also induced. At PHERES1, maternal imprinting of the promoter was disrupted, and paternal imprinting of MEDEA appeared to be lost. The rate of hybrid seed lethality was sensitive to parental genome dosage, and gene activation was proportional to the dosage of parental genomes. A causal link was established between PHE1 and hybrid seed failure; a transposon-induced disruption of PHE1 significantly improved fertility. We propose that the dosage-dependent regulation of chromatin could be a universal phenomenon affecting lethality in interspecies hybrids.

Arabidopsis↗
PubMed2006

The Arabidopsis thaliana transcriptome in response to Agrobacterium tumefaciens.

The pathogen Agrobacterium tumefaciens infects a broad range of plants, introducing the T-DNA into their genome. Contrary to all known bacterial phytopathogens, A. tumefaciens lacks the hypersensitive response-inducing hrp genes, although it introduces numerous proteins into the plant cell through a type IV secretion system. To understand the timing and extent of the plant transcriptional response to this unusual pathogen, we used an Arabidopsis 26,000-gene oligonucleotide microarray. We inoculated Arabidopsis cell cultures with an oncogenic Agrobacterium strain and analyzed four biological replicates to identify two robust sets of regulated genes, one induced and the other suppressed. In both cases, the response was distinct at 48 h after infection, but not at 24 h or earlier. The induced set includes genes encoding known defense proteins, and the repressed set is enriched with genes characteristic of cell proliferation even though a growth arrest was not visible in the inoculated cultures. The analysis of the repressed genes revealed that the conserved upstream regulatory elements Frankiebox (also known as "site II") and Telobox are associated with the suppression of gene expression. The regulated gene sets should be useful in dissecting the signaling pathways in this plant-pathogen interaction.

Agrobacterium tumefaciens↗
PubMed2006

TILLING: practical single-nucleotide mutation discovery.

In the post-genomic sequencing era, an expanding portfolio of genomic technologies has been applied to the study of gene function. Reverse genetics approaches that provide targeted inactivation of genes identified by sequence analysis include TILLING (for Targeting Local Lesions IN Genomes). TILLING searches the genomes of mutagenized organisms for mutations in a chosen gene, typically single base-pair substitutions. This review covers practical aspects of the technology, ranging from building the mutagenized population to mutation discovery, and discusses possible improvements to current protocols and the impact of new genomic methods for mutation discovery in relation to the future of the TILLING approach.

Mutation↗
PubMed2006

High-throughput TILLING for Arabidopsis.

Targeting induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. In this chapter, we describe the basic methodology for high-throughput TILLING. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated sequence and its wild-type counterpart. These heteroduplexes are substrates for cleavage by the endonuclease CEL I. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. High-throughput TILLING has been adopted by the Arabidopsis TILLING Project (ATP) to provide allelic series of point mutations for the general Arabidopsis community.

Arabidopsis↗
PubMed2005

A conserved and species-specific functional interaction between the Werner syndrome-like exonuclease atWEX and the Ku heterodimer in Arabidopsis.

Werner syndrome is associated with mutations in the DNA helicase RecQ3 [a.k.a. Homo sapiens (hs)WRN]. The function of hsWRN is unknown although biochemical studies suggest a role in DNA ends stability and repair. Unlike other RecQ family members, hsWRN possesses an N-terminal domain with exonuclease activity, which is stimulated by interaction with the Ku heterodimer. While this interaction is intriguing, we do not know whether it is important for hsWRN function. Although flies, worms, fungi and plants do not have RecQ-like (RQL) helicases with an intrinsic exonuclease activity, they possess proteins having domains homologous to the hsWRN exonuclease. The genome of Arabidopsis thaliana (at) encodes multiple RQL and a single protein with homology to the WRN exonuclease domain, atWEX (Werner-like Exonuclease). Here we show that atWEX has properties that are similar to hsWRN. atWEX binds to and is stimulated by atKu. Interestingly, stimulation by Ku is species-specific, as hsKu does not stimulate atWEX exonuclease activity. Likewise, atKu fails to enhance the exonuclease activity of hsWRN. Thus, in spite of the differences in structural organization, the functional interaction between WRN-like exonucleases and Ku has been preserved through evolutionary radiation of species, emphasizing the importance of this interaction in cell function.

Amino Acid Sequence↗
PubMed2005

Genomewide nonadditive gene regulation in Arabidopsis allotetraploids.

Polyploidy has occurred throughout the evolutionary history of all eukaryotes and is extremely common in plants. Reunification of the evolutionarily divergent genomes in allopolyploids creates regulatory incompatibilities that must be reconciled. Here we report genomewide gene expression analysis of Arabidopsis synthetic allotetraploids, using spotted 70-mer oligo-gene microarrays. We detected >15% transcriptome divergence between the progenitors, and 2105 and 1818 genes were highly expressed in Arabidopsis thaliana and A. arenosa, respectively. Approximately 5.2% (1362) and 5.6% (1469) genes displayed expression divergence from the midparent value (MPV) in two independently derived synthetic allotetraploids, suggesting nonadditive gene regulation following interspecific hybridization. Remarkably, the majority of nonadditively expressed genes in the allotetraploids also display expression changes between the parents, indicating that transcriptome divergence is reconciled during allopolyploid formation. Moreover, >65% of the nonadditively expressed genes in the allotetraploids are repressed, and >94% of the repressed genes in the allotetraploids match the genes that are expressed at higher levels in A. thaliana than in A. arenosa, consistent with the silencing of A. thaliana rRNA genes subjected to nucleolar dominance and with overall suppression of the A. thaliana phenotype in the synthetic allotetraploids and natural A. suecica. The nonadditive gene regulation is involved in various biological pathways, and the changes in gene expression are developmentally regulated. In contrast to the small effects of genome doubling on gene regulation in autotetraploids, the combination of two divergent genomes in allotetraploids by interspecific hybridization induces genomewide nonadditive gene regulation, providing a molecular basis for de novo variation and allopolyploid evolution.

Arabidopsis↗
PubMed2005

The plant cell defense and Agrobacterium tumefaciens.

We previously identified changes in gene expression in Ageratum conyzoides plant cells inoculated with Agrobacterium tumefaciens by using cDNA-AFLP. Here, we show that a subset of defense-related genes is differentially regulated by an Agrobacterium attachment-deficient mutant. The expression pattern triggered by this mutant is similar to that induced by inoculation with non-pathogenic bacteria. We also observed that the expression level of the defense genes was inversely correlated with the efficiency of transformation by Agrobacterium. We propose that the plant defense system has an important role in controlling infection and transformation and that Agrobacterium may dampen some plant defense responses.

Ageratum↗
PubMed2005

Aneuploidy and genetic variation in the Arabidopsis thaliana triploid response.

Polyploidy, the inheritance of more than two genome copies per cell, has played a major role in the evolution of higher plants. Little is known about the transition from diploidy to polyploidy but in some species, triploids are thought to function as intermediates in this transition. In contrast, in other species triploidy is viewed as a block. We investigated the responses of Arabidopsis thaliana to triploidy. The role of genetic variability was tested by comparing triploids generated from crosses between Col-0, a diploid, and either a natural autotetraploid (Wa-1) or an induced tetraploid of Col-0. In this study, we demonstrate that triploids of A. thaliana are fertile, producing a swarm of different aneuploids. Propagation of the progeny of a triploid for a few generations resulted in diploid and tetraploid cohorts. This demonstrated that, in A. thaliana, triploids can readily form tetraploids and function as bridges between euploid types. Genetic analysis of recombinant inbred lines produced from a triploid identified a locus on chromosome I exhibiting allelic bias in the tetraploid lines but not in the diploid lines. Thus, genetic variation was subject to selection contingent on the final ploidy and possibly acting during the protracted aneuploid phase.

Alleles↗
PubMed2005

Two callose synthases, GSL1 and GSL5, play an essential and redundant role in plant and pollen development and in fertility.

Callose, a beta-1,3-glucan that is widespread in plants, is synthesized by callose synthase. Arabidopsis thaliana contains a family of 12 putative callose synthase genes (GSL1-12). The role of callose and of the individual genes in plant development is still largely uncertain. We have now used TILLING and T-DNA insertion mutants (gsl1-1, gsl5-2 and gsl5-3) to study the role of two closely related and linked genes, GSL1 and GSL5, in sporophytic development and in reproduction. Both genes are expressed in all parts of the plant. Sporophytic development was nearly normal in gsl1-1 homozygotes and only moderately defective in homozygotes for either of the two gsl5 alleles. On the other hand, plants that were gsl1-1/+ gsl5/gsl5 were severely defective, with smaller leaves, shorter roots and bolts and smaller flowers. Plants were fertile when the sporophytes had either two wild-type GSL1 alleles, or one GSL5 allele in a gsl1-1 background, but gsl1-1/+ gsl5/gsl5 plants produced an extremely reduced number of viable seeds. A chromosome with mutations in both GSL1 and GSL5 rendered pollen infertile, although such a chromosome could be transmitted via the egg. As a result, it was not possible to obtain plants that were homozygous for mutations in both the GSL genes. Pollen grain development was severely affected in double mutant plants. Many pollen grains were collapsed and inviable in the gsl1-1/gsl1-1 gsl5/+ and gsl1-1/+ gsl5/gsl5 plants. In addition, gsl1-1/+ gsl5/gsl5 plants produced abnormally large pollen with unusual pore structures, and had problems with tetrad dissociation. In this particular genotype, while the callose wall formed around the pollen mother cells, no callose wall separated the resulting tetrads. We conclude that GSL1 and GSL5 play important, but at least partially redundant roles in both sporophytic development and in the development of pollen. They are responsible for the formation of the callose wall that separates the microspores of the tetrad, and also play a gametophytic role later in pollen grain maturation. Other GSL genes may control callose formation at different steps during pollen development.

Arabidopsis↗
PubMed2005

Methods for genome-wide analysis of gene expression changes in polyploids.

Polyploidy is an evolutionary innovation, providing extra sets of genetic material for phenotypic variation and adaptation. It is predicted that changes of gene expression by genetic and epigenetic mechanisms are responsible for novel variation in nascent and established polyploids (Liu and Wendel, 2002; Osborn et al., 2003; Pikaard, 2001). Studying gene expression changes in allopolyploids is more complicated than in autopolyploids, because allopolyploids contain more than two sets of genomes originating from divergent, but related, species. Here we describe two methods that are applicable to the genome-wide analysis of gene expression differences resulting from genome duplication in autopolyploids or interactions between homoeologous genomes in allopolyploids. First, we describe an amplified fragment length polymorphism (AFLP)--complementary DNA (cDNA) display method that allows the discrimination of homoeologous loci based on restriction polymorphisms between the progenitors. Second, we describe microarray analyses that can be used to compare gene expression differences between the allopolyploids and respective progenitors using appropriate experimental design and statistical analysis. We demonstrate the utility of these two complementary methods and discuss the pros and cons of using the methods to analyze gene expression changes in autopolyploids and allopolyploids. Furthermore, we describe these methods in general terms to be of wider applicability for comparative gene expression in a variety of evolutionary, genetic, biological, and physiological contexts.

Arabidopsis↗
PubMed2005

The advantages and disadvantages of being polyploid.

Polyploids - organisms that have multiple sets of chromosomes - are common in certain plant and animal taxa, and can be surprisingly stable. The evidence that has emerged from genome analyses also indicates that many other eukaryotic genomes have a polyploid ancestry, suggesting that both humans and most other eukaryotes have either benefited from or endured polyploidy. Studies of polyploids soon after their formation have revealed genetic and epigenetic interactions between redundant genes. These interactions can be related to the phenotypes and evolutionary fates of polyploids. Here, I consider the advantages and challenges of polyploidy, and its evolutionary potential.

Evolution, Molecular↗
PubMed2005

Genomic changes in synthetic Arabidopsis polyploids.

Polyploids are common and arise frequently by genome duplication (autopolyploids) or interspecific hybridization (allopolyploids). Neoallopolyploids display sterility, lethality, phenotypic instability, gene silencing and epigenetic changes. Little is known about the molecular basis of these phenomena, and how much genomic remodeling happens upon allopolyploidization. Extensive genomic remodeling has been documented in wheat, but little remodeling occurs in cotton. Newly synthesized Arabidopsis allopolyploids, which display phenotypic instability and low fertility, displayed several, possibly related mechanisms that can remodel genomes. We detected transcriptional activity of several transposons although their transposition was limited. One represents a new family of conditionally active En-Spm-like transposons of Arabidopsis thaliana, which underwent remodeling of CG methylation upon allopolyploidization. A random amplified fragment length polymorphism survey suggested remodeling at few, specific loci. Meiotic analyses revealed the appearance of chromosomal fragments in a substantial fraction of anther meiocytes. In several individuals produced by hybrids between the synthetic and a natural allopolyploid pollen viability inversely correlated with meiotic instability. Activity of selected DNA transposons and the possibly related chromosomal breaks could cause changes by inducing translocations and rearrangements.

Arabidopsis↗
PubMed2004

Chromosomal locus rearrangements are a rapid response to formation of the allotetraploid Arabidopsis suecica genome.

Allopolyploidy is a significant evolutionary process, resulting in new species with diploid or greater chromosome complements derived from two or more progenitor species. We examined the chromosomal consequences of genomic merger in Arabidopsis suecica, the allotetraploid hybrid of Arabidopsis thaliana and Arabidopsis arenosa. Fluorescence in situ hybridization with centromere, nucleolus organizer region (NOR), and 5S rRNA gene probes reveals the expected numbers of progenitor chromosomes in natural A. suecica, but one pair of A. thaliana NORs and one pair of A. arenosa-derived 5S gene loci are missing. Similarly, in newly formed synthetic A. suecica-like allotetraploids, pairs of A. thaliana NORs are gained de novo, lost, and/or transposed to A. arenosa chromosomes, with genotypic differences apparent between F(3) siblings of the same F(2) parent and between independent lines. Likewise, pairs of A. arenosa 5S genes are lost and novel linkages between 5S loci and NORs arise in synthetic allotetraploids. By contrast, the expected numbers of A. arenosa-derived NORs and A. thaliana-derived 5S loci are found in both natural and synthetic A. suecica. Collectively, these observations suggest that some, but not all, loci are unstable in newly formed A. suecica allotetraploids and can participate in a variety of alternative rearrangements, some of which resemble chromosomal changes found in nature.

Arabidopsis↗
PubMed2004

The effect of stress on genome regulation and structure.

BACKGROUND: Stresses exert evolutionary pressures on all organisms, which have developed sophisticated responses to cope and survive. These responses involve cellular physiology, gene regulation and genome remodelling. SCOPE: In this review, the effects of stress on genomes and the connected responses are considered. Recent developments in our understanding of epigenetic genome regulation, including the role of RNA interference (RNAi), suggest a function for this in stress initiation and response. We review our knowledge of how different stresses, tissue culture, pathogen attack, abiotic stress, and hybridization, affect genomes. Using allopolyploid hybridization as an example, we examine mechanisms that may mediate genomic responses, focusing on RNAi-mediated perturbations. CONCLUSIONS: A common response to stresses may be the relaxation of epigenetic regulation, leading to activation of suppressed sequences and secondary effects as regulatory systems attempt to re-establish genomic order.

Climate↗
PubMed2004

Discovery of induced point mutations in maize genes by TILLING.

BACKGROUND: Going from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community. RESULTS: We demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious. CONCLUSIONS: Our findings indicate that TILLING is a broadly applicable and efficient reverse-genetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project.

Ethyl Methanesulfonate↗