A complete BAC-based physical map of the Arabidopsis thaliana genome.

Arabidopsis thaliana is a small flowering plant that serves as the major model system in plant molecular genetics. The efforts of many scientists have produced genetic maps that provide extensive coverage of the genome (http://genome-www. stanford.edu/Arabidopsis/maps.html). Recently, detailed YAC, BAC, P1 and cosmid-based physical maps (that is, representations of genomic regions as sets of overlapping clones of corresponding libraries) have been established that extend over wide genomic areas ranging from several hundreds of kilobases to entire chromosomes. These maps provide an entry to gain deeper insight into the A. thaliana genome structure. A. thaliana has been chosen as the subject of the first large-scale project intended to determine the full genome sequence of a plant. This sequencing project, together with the increasing interest in map-based gene cloning, has highlighted the requirement for a complete and accurate physical map of this plant species. To supply the scientific community with a high-quality resource, we present here a complete physical map of A. thaliana using essentially the IGF BAC library. The map consists of 27 contigs that cover the entire genome, except for the presumptive centromeric regions, nucleolar organization regions (NOR) and telomeric areas. This is the first reported map of a complex organism based entirely on BAC clones and it represents the most homogeneous and complete physical map established to date for any plant genome. Furthermore, the analysis performed here serves as a model for an efficient physical mapping procedure using BAC clones that can be applied to other complex genomes.

The ethylene signal transduction pathway in plants.

Ethylene (C2H4), the chemically simplest plant hormone, is among the best-characterized plant growth regulators. It participates in a variety of stress responses and developmental processes. Genetic studies in Arabidopsis have defined a number of genes in the ethylene signal transduction pathway. Isolation of two of these genes has revealed that plants sense this gas through a combination of proteins that resemble both prokaryotic and eukaryotic signaling proteins. Ethylene signaling components are likely conserved for responses as diverse as cell elongation, cell fate patterning in the root epidermis, and fruit ripening. Genetic manipulation of these genes will provide agriculture with new tools to prevent or modify ethylene responses in a variety of plants.

EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis.

Ethylene regulates plant growth, development, and responsiveness to a variety of stresses. Cloning of the Arabidopsis EIN2 gene identifies a central component of the ethylene signaling pathway. The amino-terminal integral membrane domain of EIN2 shows similarity to the disease-related Nramp family of metal-ion transporters. Expression of the EIN2 CEND is sufficient to constitutively activate ethylene responses and restores responsiveness to jasmonic acid and paraquat-induced oxygen radicals to mutant plants. EIN2 is thus recognized as a molecular link between previously distinct hormone response pathways. Plants may use a combinatorial mechanism for assessing various stresses by enlisting a common set of signaling molecules.

Ethylene gas: it's not just for ripening any more!

Hormones play a central role in regulating plant growth and development. There are five well-characterized plant hormones, the chemically simplest of which is the gaseous olefin ethylene. Molecular genetic studies of Arabidopsis are beginning to reveal the mechanisms controlling ethylene biosynthesis, perception and signal transduction.

Ethylene signaling: from mutants to molecules.

The past decade has been incredibly productive for ethylene researchers. Major components in the ethylene signaling pathway in plants have been identified and characterized. The past year's contributions include the crystallographic analysis of the Arabidopsis ETR1 receiver domain, antisense studies of the tomato ethylene receptor genes LeETR4 and NR, and the cloning and functional characterization of several Arabidopsis EREBP-related transcription activators and repressors, and of an EIN3-ortholog of tobacco. Additional evidence for the interconnection of the ethylene and auxin responses was provided by the cloning and characterization of Arabidopsis NPH4. Finally, the first discovery of ethylene responsiveness in an animal species implied a more universal role for ethylene than previously thought.

Ethylene gas: perception, signaling and response.

During the last decade a genetic approach based on the Arabidopsis 'triple response' to the hormone ethylene has allowed the identification of numerous components of the signal transduction pathway. Cloning of the genes and biochemical analysis of the proteins that they encode are uncovering the molecular mechanisms that allow a plant cell to perceive and respond to this gaseous regulator of plant growth/stress responses.

Human DNA methylomes of neurodegenerative diseases show common epigenomic patterns.

Different neurodegenerative disorders often show similar lesions, such as the presence of amyloid plaques, TAU-neurotangles and synuclein inclusions. The genetically inherited forms are rare, so we wondered whether shared epigenetic aberrations, such as those affecting DNA methylation, might also exist. The studied samples were gray matter samples from the prefrontal cortex of control and neurodegenerative disease-associated cases. We performed the DNA methylation analyses of Alzheimer's disease, dementia with Lewy bodies, Parkinson's disease and Alzheimer-like neurodegenerative profile associated with Down's syndrome samples. The DNA methylation landscapes obtained show that neurodegenerative diseases share similar aberrant CpG methylation shifts targeting a defined gene set. Our findings suggest that neurodegenerative disorders might have similar pathogenetic mechanisms that subsequently evolve into different clinical entities. The identified aberrant DNA methylation changes can be used as biomarkers of the disorders and as potential new targets for the development of new therapies.

Genetic analysis of ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway.

The response of Arabidopsis thaliana etiolated seedlings to the plant hormone ethylene is a conspicuous phenotype known as the triple response. We have identified genes that are required for ethylene perception and responses by isolating mutants that fail to display a triple response in the presence of exogenous ethylene. Five new complementation groups have been identified. Four of these loci, designated ein4, ein5, ein6 and ein7, are insensitive to ethylene. The fifth complementation group, eir1, is defined by a novel class of mutants that have agravitropic and ethylene-insensitive roots. Double-mutant phenotypes have allowed the positioning of these loci in a genetic pathway for ethylene signal transduction. The ethylene-response pathway is defined by the following loci: ETR1, EIN4, CTR1, EIN2, EIN3, EIN5, EIN6, EIN7, EIR1, AUX1 and HLS1. ctr1-1 is epistatic to etr1-3 and ein4, indicating that CTR1 acts after both ETR1 and EIN4 in the ethylene-response pathway. Mutations at the EIN2, EIN3, EIN5, EIN6 and EIN7 loci are all epistatic to the ctr1 seedling phenotype. The EIR1 and AUX1 loci define a root-specific ethylene response that does not require EIN3 or EIN5 gene activity. HLS1 appears to be required for differential cell growth in the apical hook. The EIR1, AUX1 and HLS1 genes may function in the interactions between ethylene and other plant hormones that occur late in the signaling pathway of this simple gas.

The ethylene pathway: a paradigm for plant hormone signaling and interaction.

To dissect the web of signals that control plant growth, it is important to understand how the individual components of the pathway are modulated. Ethylene is a plant hormone involved in a large number of developmental processes. Biochemical and genetic approaches have provided a detailed view of the biosynthetic and signal transduction pathways of this hormone in the reference plant Arabidopsis thaliana. The effects of several hormones and of developmental changes on the regulation of the key enzymes of ethylene biosynthesis, ACC synthase and ACC oxidase, serve as a clear example of interaction between signals in the generation of complex responses. We now have a picture of how ethylene is sensed by the ethylene receptors and how the signal is further transduced to the nucleus. Although some of the ethylene receptors show a tissue-specific pattern of expression, little is known about the regulation of the components of the ethylene transduction cascade by other hormones or developmental factors. Once the ethylene signal reaches the nucleus, it activates a transcriptional cascade that results in changes in the expression of a number of genes. We describe some of the results that suggest an interaction at the transcriptional level between ethylene, other hormones, and stress signals.

Disease development in ethylene-insensitive Arabidopsis thaliana infected with virulent and avirulent Pseudomonas and Xanthomonas pathogens.

The plant hormone ethylene has been hypothesized to play roles both in disease resistance and in disease susceptibility. These processes were examined by using isogenic virulent and avirulent bacterial pathogens and mutants of Arabidopsis thaliana that were altered in ethylene physiology. Ethylene-insensitive ein1 and ein2 mutants of Arabidopsis were resistant to Pseudomonas syringae pv. tomato made avirulent by the addition of the cloned avirulence genes avrRpt2, avrRpm1, or avrB; this suggests that ethylene is not required for active resistance against avirulent bacteria. In a second set of experiments, susceptibility was monitored with virulent P. s. pv. tomato, P. s. pv. maculicola, or Xanthomonas campestris pv. campestris strains. Wild-type Arabidopsis and ein1 mutants were susceptible to these strains, but ein2 mutants developed only minimal disease symptoms. Despite these reduced symptoms, virulent P. s. pv. tomato grew extensively within ein2 leaves. The Pseudomonas phytotoxin coronatine induces ethylene biosynthesis and diseaselike symptoms on many plant species, but the reduced symptomology of ein2 mutants could not be attributed to insensitivity to coronatine. The enhanced disease tolerance of ein2 plants suggests that ethylene may mediate pathogen-induced damage, but the absence of tolerance in ein1 mutants has yet to be explained.

Epiallelic variation in Arabidopsis thaliana.

Genotype is the primary determinate of phenotype. During the past two decades, however, there has been an emergent recognition of the epigenotype, a separate layer of heredity distinct from the primary DNA sequence that can have profound effects on phenotype. The epigenotype is a collection of chemical modifications to the DNA and nucleosomes in conjunction with noncoding RNA transcripts, and together these epigenetic marks act as a potent and expansive regulatory system for controlling gene expression. In this review, we discuss our current understanding of variation in epigenotype in the model plant Arabidopsis and how allelic differences attributable to epigenetic changes, or epialleles, can affect phenotype. We discuss examples of epialleles that have been created in the laboratory and others that have been identified in natural populations, because these two models provide complementary information regarding the genetic pathways, mechanisms of transmission, and biological and evolutionary context for the role of the epigenotype in phenotypic variation.

Genome-wide analysis of chromosomal features repressing human immunodeficiency virus transcription.

We have investigated regulatory sequences in noncoding human DNA that are associated with repression of an integrated human immunodeficiency virus type 1 (HIV-1) promoter. HIV-1 integration results in the formation of precise and homogeneous junctions between viral and host DNA, but integration takes place at many locations. Thus, the variation in HIV-1 gene expression at different integration sites reports the activity of regulatory sequences at nearby chromosomal positions. Negative regulation of HIV transcription is of particular interest because of its association with maintaining HIV in a latent state in cells from infected patients. To identify chromosomal regulators of HIV transcription, we infected Jurkat T cells with an HIV-based vector transducing green fluorescent protein (GFP) and separated cells into populations containing well-expressed (GFP-positive) or poorly expressed (GFP-negative) proviruses. We then determined the chromosomal locations of the two classes by sequencing 971 junctions between viral and cellular DNA. Possible effects of endogenous cellular transcription were characterized by transcriptional profiling. Low-level GFP expression correlated with integration in (i) gene deserts, (ii) centromeric heterochromatin, and (iii) very highly expressed cellular genes. These data provide a genome-wide picture of chromosomal features that repress transcription and suggest models for transcriptional latency in cells from HIV-infected patients.

Exploiting the triple response of Arabidopsis to identify ethylene-related mutants.

Alterations in the response of dark-grown seedlings to ethylene (the "triple response") were used to isolate a collection of ethylene-related mutants in Arabidopsis thaliana. Mutants displaying a constitutive response (eto1) were found to produce at least 40 times more ethylene than the wild type. The morphological defects in etiolated eto1-1 seedlings reverted to wild type under conditions in which ethylene biosynthesis or ethylene action were inhibited. Mutants that failed to display the apical hook in the absence of ethylene (his1) exhibited reduced ethylene production. In the presence of exogenous ethylene, hypocotyl and root of etiolated his1-1 seedlings were inhibited in elongation but no apical hook was observed. Mutants that were insensitive to ethylene (ein1 and ein2) produced increased amounts of ethylene, displayed hormone insensitivity in both hypocotyl and root responses, and showed an apical hook. Each of the "triple response" mutants has an effect on the shape of the seedling and on the production of the hormone. These mutants should prove to be useful tools for dissecting the mode of ethylene action in plants.

Development of large DNA methods for plants: molecular cloning of large segments of Arabidopsis and carrot DNA into yeast.

Procedures for the preparation, analysis and cloning of large DNA molecules from two different plant species are described. Arabidopsis and carrot protoplasts were used for the preparation of large DNA molecules in agarose "plugs" or in solution. Pulsed-field gel electrophoresis (PFGE) analysis of large plant DNA preparations using a contour-clamped homogeneous field (CHEF) apparatus indicated that the size of the DNA was at least 12 Mb. Large DNA preparations were shown to be useful for restriction enzyme analysis of the Arabidopsis genome using both frequent and infrequent cutting enzymes and for the molecular cloning of large segments of DNA into yeast using artificial chromosome (YAC) vectors. PFGE and blot hybridization analysis of Arabidopsis and carrot DNA-containing YACs indicated that both unique and highly repeated DNA sequences were represented in these libraries.

Inhibition of gene expression in plant cells by expression of antisense RNA.

Due to the paucity of mutations in biochemical pathways in plants, an alternative approach to classical genetics was tested by expressing antisense RNA in plant cells. A series of plasmids was constructed with the bacterial gene for chloramphenicol acetyltransferase (EC 2.3.1.28) linked in either the sense or antisense orientation to several different plant gene promoters. Various ratios of sense and antisense chloramphenicol acetyltransferase gene plasmids were introduced into plant protoplasts by electric field-mediated DNA transfer ("electroporation") and the level of expression in each combination was monitored by chloramphenicol acetyltransferase assays. Transcription of antisense RNA was found to effectively block the expression of target genes. Thus, the observation that antisense RNA inhibits gene expression in bacteria and animal systems has been extended to the plant kingdom. Antisense RNA techniques have immediate practical applications in both basic research and in plant genetic engineering.

Plant defense genes are regulated by ethylene.

One of the earliest detectable events during plant-pathogen interaction is a rapid increase in ethylene biosynthesis. This gaseous plant stress hormone may be a signal for plants to activate defense mechanisms against invading pathogens such as bacteria, fungi, and viruses. The effect of ethylene on four plant genes involved in three separate plant defense response pathways was examined; these included (i and ii) genes that encode L-phenylalanine ammonia-lyase (EC 4.3.1.5) and 4-coumarate:CoA ligase [4-coumarate:CoA ligase (AMP-forming), EC 6.2.1.12], enzymes of the phenylpropanoid pathway, (iii) the gene encoding chalcone synthase, an enzyme of the flavonoid glycoside pathway, and (iv) the genes encoding hydroxyproline-rich glycoprotein, a major protein component(s) of plant cell walls. Blot hybridization analysis of mRNA from ethylene-treated carrot roots reveals marked increases in the levels of phenylalanine ammonia-lyase mRNA, 4-coumarate CoA ligase mRNA, chalcone synthase mRNA, and certain hydroxyproline-rich glycoprotein transcripts. The effect of ethylene on hydroxyproline-rich glycoprotein mRNA accumulation was different from that of wounding. Ethylene induces two hydroxyproline-rich glycoprotein mRNAs (1.8 and 4.0 kilobases), whereas wounding of carrot root leads to accumulation of an additional hydroxyproline-rich mRNA (1.5 kilobases). These results indicate that at least two distinct signals, ethylene and a wound signal, can affect the expression of plant defense-response genes.

Assignment of 30 microsatellite loci to the linkage map of Arabidopsis.

Thirty microsatellite loci were assigned to the Arabidopsis linkage map. Several microsatellite sequences in Arabidopsis DNA were found by searching the EMBL and GenBank databases, and a number of these were subsequently found to detect polymorphisms between different Arabidopsis strains by the polymerase chain reaction (PCR). After the presence of microsatellites in Arabidopsis and their utility for genetic mapping had been demonstrated, systematic screening for (CA)n and (GA)n sequences was carried out on marker-selected plasmid libraries and a small-insert genomic library. Positive clones were sequenced, PCR primers flanking the repeats were synthesized, and PCR was carried out on different strains to look for useful polymorphisms. Surprisingly, of 18 (CA)n repeats (n > 13), only one was polymorphic. In contrast, 25 of 30 (GA)n repeats, 2 of 3 (AT)n repeats, and 2 of 4 (A)n repeats were polymorphic. The majority of the (CA)n repeats were complex, with adjacent short di-, tri-, or tetranucleotide repeats, whereas most of the (GA)n, (TA)n, and (A)n repeats were simple. The (CA)n repeats were also refractory to PCR analysis, requiring extensive optimization of PCR conditions, whereas the other repeat classes were mostly amplified with a single set of standard conditions. When polymorphisms were detected, the microsatellites were mapped using a set of recombinant inbred lines originating from a cross between the strains Columbia and Landsberg erecta.

Genetic analysis of a seedling stress response to ethylene in Arabidopsis.

A genetic framework has been devised for the action of genes within the ethylene-response pathway. This working model is based on the epistatic interactions among a variety of ethylene response mutations. Most of the mutations that have been described act in a linear pathway. Genes controlling cell elongation in response to ethylene must, at some level, act to affect the architecture of the cytoskeleton. Genes that act late in the pathway, in mutant form, may lead to highly specific phenotypes such as the increased sensitivity to taxol in the ein6 mutant. Analysis of these downstream components may provide critical insights into the nature of ethylene's effect on the cell elongation machinery.

Varicella-zoster virus vaccine DNA differs from the parental virus DNA.

The DNAs of a varicella-zoster virus vaccine and its parental virus were compared by CsCl buoyant density centrifugation and restriction enzyme cleavage analysis. The varicella-zoster virus vaccine DNA showed a heterogeneous buoyant profile and altered restriction enzyme cleavage patterns. These changed properties are probably the result of the accumulation of virus containing defective varicella-zoster virus DNA during extensive cell culture passage of the vaccine virus.

Variables that may influence the alkaline sedimentation pattern of herpes simplex virus DNA.

Herpes simplex virus (HSV) DNA sediments homogeneously on a neutral sucrose gradient but heterogeneously on an alkaline sucrose gradient. Several factors that may influence the alkaline sedimentation pattern of HSV DNA were examined: e.g., the host cell, cell density at time of infection, multiplicity of infection, and the starting material for HSV DNA purification (whether HSV-infected cells or cell-free virus). Based on alkaline sedimentation analysis, these factors appear to play little or no role in the amount of intact single-stranded HSV DNA observed.