Clearing a path through the jungle: progress in Arabidopsis genomics

Bevan, Michael W.; Bancroft, Ian; Mewes, Hans‐Werner; Martienssen, Rob; McCombie, Richard W.

doi:10.1002/(sici)1521-1878(199902)21:2<110::aid-bies5>3.0.co;2-v

Cited by 27 publications

(11 citation statements)

References 51 publications

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“…Sixty-two CIC clones out of 1152 were positive with the TSI-A probe. Twenty-six of these also contained the pericentromeric 180-bp repeat (Martinez-Zapater et al, 1986;Maluszynka and HeslopHarrison, 1991), seven contained 5S RNA genes known to be located in the vicinity of a centromere (Bevan et al, 1999), and 16 clones contained other markers that mapped close to centromeres. Only four positive clones mapped outside of centromeric regions.…”

Section: Organization Of Chromosomal Tsi Templatesmentioning

confidence: 99%

Endogenous Targets of Transcriptional Gene Silencing in Arabidopsis

et al. 2000

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Transcriptional gene silencing (TGS) frequently inactivates foreign genes integrated into plant genomes but very likely also suppresses an unknown subset of chromosomal information. Accordingly, RNA analysis of mutants impaired in silencing should uncover endogenous targets of this epigenetic regulation. We compared transcripts from wild-type Arabidopsis carrying a silent transgene with RNA from an isogenic transgene-expressing TGS mutant. Two cDNA clones were identified representing endogenous RNA expressed only in the mutant. The synthesis of these RNAs was found to be released in several mutants affected in TGS, implying that TGS in general and not a particular mutation controls the transcriptional activity of their templates. Detailed analysis revealed that the two clones are part of longer transcripts termed TSI (for transcriptionally silent information). Two major classes of related TSI transcripts were found in a mutant cDNA library. They are synthesized from repeats present in heterochromatic pericentromeric regions of Arabidopsis chromosomes. These repeats share sequence homology with the 3 Ј terminal part of the putative retrotransposon Athila . However, the transcriptional activation does not include the transposon itself and does not promote its movement. There is no evidence for a general release of silencing from retroelements. Thus, foreign genes in plants encounter the epigenetic control normally directed, at least in part, toward a subset of pericentromeric repeats. INTRODUCTIONAn appropriate balance between activation and repression of genetic information is intrinsic to any living cell. Tight control of gene expression is necessary for adaptation to environmental factors, regulation of physiological parameters, and development of specialized cell types within a multicellular organism. Differentiation, in particular, is the result of mitotically heritable changes in gene expression, where the acquired states of gene activity reach a certain epigenetic stability. This stability arises by strict control of gene activators, by regulation of transcript stability, by altering the transcriptional availability of the genetic information itself, or by some combination of these. Alteration of the transcriptional availability of genetic information may be based on stable epigenetic silencing of selected genetic loci. This type of silencing is also evident from unexpected repression of transgene activities in various experimental systems (reviewed in Depicker and Van Montagu, 1997;Stam et al., 1997;Henikoff, 1998;Vaucheret et al., 1998;Grant, 1999).The silencing of transgenic loci has received particular attention in plants, because it reduces the reliability of transgenic approaches in biotechnology. Complex inserts containing rearranged multiple copies of a foreign DNA are particularly prone to gene silencing. Loss of transgene expression may occur by two different mechanisms. The first precludes transcription (transcriptional gene silencing, or TGS); the second targets selected transcripts for rapid de...

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Section: Organization Of Chromosomal Tsi Templatesmentioning

confidence: 99%

Endogenous Targets of Transcriptional Gene Silencing in Arabidopsis

et al. 2000

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“…An antisense cDNA library of Arabidopsis in this vector was generated using mRNA from mature rosette leaf tissue of Arabidopsis. Arabidopsis was chosen for the construction of the antisense cDNA library because the whole genome has been sequenced in this organism (Meinke et al 1998;Bevan et al 1999; The Arabidopsis Genome Initiative 2000). The genome sequence database allows easy identification of a whole gene sequence once a partial sequence of the cDNA molecule for a given mutant phenotype is obtained.…”

Section: Construction Of the Antisense Cdna Librarymentioning

confidence: 99%

Random antisense cDNA mutagenesis as an efficient functional genomic approach in higher plants

Jun

Kim

Cho

et al. 2002

Planta

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Most cellular processes in an organism depend on functions of expressed sequences. Thus, efficient large-scale functional assignment of expressed sequences is crucial for understanding cellular processes. Towards this goal in plants, we designed a "random antisense cDNA mutagenesis (RAM)" approach. In a pilot experiment, 1,000 transgenic plants of Arabidopsis thaliana (L.) Heynh. (ecotype Wassilevskija) expressing random antisense cDNA(s) were generated from Agrobacterium cultures harboring an Arabidopsis antisense cDNA library. We identified 104 mutant lines from the transgenic pool by visual screening. Genetic analysis suggested that 37% of the mutations were likely due to antisense effects. When the cDNA inserts were isolated from 11 mutant lines by polymerase chain reaction and reintroduced into plants to express the antisense transcripts, the original mutant phenotypes were reproduced in 7 cDNA clones. One of the cDNA clones did not generate a database match to any sequence with known functions, but did have a dramatic effect on the architecture of the inflorescence in the antisense transgenic plants. Through the RAM approach, it should be possible to assign a large number of expressed sequences to known in vivo functions in plants.

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“…Indeed, with the current availability of Arabidopsis genome sequences from public databases, half of all potential insertions can be matched to sequenced regions of the genome and placed on a physical map. Insertional mutagenesis is especially effective for generating gene knockouts in Arabidopsis because of the high gene density (approximately one gene every 5 kb; Bevan et al, 1998Bevan et al, , 1999, which means that on average, one out of two insertions results in gene disruption.…”

Section: Introductionmentioning

confidence: 99%

Analysis of Flanking Sequences fromDissociationInsertion Lines: A Database for Reverse Genetics in Arabidopsis

et al. 1999

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We have generated Dissociation ( Ds ) element insertions throughout the Arabidopsis genome as a means of random mutagenesis. Here, we present the molecular analysis of genomic sequences that flank the Ds insertions of 931 independent transposant lines. Flanking sequences from 511 lines proved to be identical or homologous to DNA or protein sequences in public databases, and disruptions within known or putative genes were indicated for 354 lines. Because a significant portion (45%) of the insertions occurred within sequences defined by GenBank BAC and P1 clones, we were able to assess the distribution of Ds insertions throughout the genome. We discovered a significant preference for Ds transposition to the regions adjacent to nucleolus organizer regions on chromosomes 2 and 4. Otherwise, the mapped insertions appeared to be evenly dispersed throughout the genome. For any given gene, insertions preferentially occurred at the 5 Ј end, although disruption was clearly possible at any intragenic position. The insertion sites of Ͼ 500 lines that could be characterized by reference to public databases are presented in a tabular format at http:// www.plantcell.org/cgi/content/full/11/12/2263/DC1. This database should be of value to researchers using reverse genetics approaches to determine gene function.

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Clearing a path through the jungle: progress in Arabidopsis genomics

Cited by 27 publications

References 51 publications

Endogenous Targets of Transcriptional Gene Silencing in Arabidopsis

Endogenous Targets of Transcriptional Gene Silencing in Arabidopsis

Random antisense cDNA mutagenesis as an efficient functional genomic approach in higher plants

Analysis of Flanking Sequences fromDissociationInsertion Lines: A Database for Reverse Genetics in Arabidopsis

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