Genome studies and molecular evolution. Commonalities, contrasts, continuity and change in plant genomes.
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Biomedical subjects
Publications and source records attributed to R Martienssen.
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Fruit morphogenesis is a process unique to flowering plants, and yet little is known about its developmental control. Following fertilization, fruits typically undergo a dramatic enlargement that is accompanied by differentiation of numerous distinct cell types. We have identified a mutation in Arabidopsis called fruitfull (ful-1), which abolishes elongation of the silique after fertilization. The ful-1 mutation is caused by the insertion of a DsE transposable enhancer trap element into the 5' untranslated leader of the AGL8 MADS-box gene. beta-glucuronidase (GUS) reporter gene expression in the enhancer trap line is observed specifically in all cell layers of the valve tissue, but not in the replum, the septum or the seeds, and faithfully mimics RNA in situ hybridization data reported previously. The lack of coordinated growth of the fruit tissues leads to crowded seeds, a failure of dehiscence and, frequently, the premature rupture of the carpel valves. The primary defect of ful-1 fruits is within the valves, whose cells fail to elongate and differentiate. Stomata, which are frequent along the epidermis of wild-type valves, are completely eliminated in the ful mutant valves. In addition to the effect on fruit development, ful cauline leaves are broader than those of wild type and show a reduction in the number of internal cell layers. These data suggest that AGL8/FUL regulates the transcription of genes required for cellular differentiation during fruit and leaf development.
The bacterial Sec and signal recognition particle (ffh-dependent) protein translocation mechanisms are conserved between prokaryotes and higher plant chloroplasts. A third translocation mechanism in chloroplasts [the proton concentration difference (DeltapH) pathway] was previously thought to be unique. The hcf106 mutation of maize disrupts the localization of proteins transported through this DeltapH pathway in isolated chloroplasts. The Hcf106 gene encodes a receptor-like thylakoid membrane protein, which shows homology to open reading frames from all completely sequenced bacterial genomes, which suggests that the DeltapH pathway has been conserved since the endosymbiotic origin of chloroplasts. Thus, the third protein translocation pathway, of which HCF106 is a component, is found in both bacteria and plants.
The recent molecular characterization of three plant genes in which mutations cause phenotypes that mimic disease responses has provided interesting new insights into cell death in plants, but many questions remain about the nature and physiological importance of the process.
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Gene trap vectors have been used in insertional mutagenesis in animal systems to clone genes with interesting patterns of expression. These vectors are designed to allow the expression of a reporter gene when the vector inserts into a transcribed region. In this paper we examine alternative splicing events that result in the expression of a GUS reporter gene carried on a Ds element which has been designed as a gene trap vector for plants. We have developed a rapid and reliable method based on PCR to study such events. Many splice donor sites were observed in the 3' Ac border. The relative frequency of utilisation of certain splice donor and acceptor sites differed between tobacco and Arabidopsis. A higher stringency of splicing was observed in Arabidopsis.
The crucifer Arabidopsis thaliana has been used widely as a model organism for the study of plant development. We describe here the development of an efficient insertional mutagenesis system in Arabidopsis that permits identification of genes by their patterns of expression during development. Transposable elements of the Ac/Ds system carrying the GUS reporter gene have been designed to act as enhancer traps or gene traps. A novel selection scheme maximizes recovery of unlinked transposition events. In this study 491 plants carrying independent transposon insertions were generated and screened for expression patterns. One-half of the enhancer trap insertions and one-quarter of the gene trap insertions displayed GUS expression in seedlings or flowers, including expression patterns specific to organs, tissues, cell types, or developmental stages. The patterns identify genes that act during organogenesis, pattern formation, or cell differentiation. Transposon insertion lines with specific GUS expression patterns provide valuable markers for studies of Arabidopsis development and identify new cell types or subtypes in plants. The diversity of gene expression patterns generated suggests that the identification and cloning of Arabidopsis genes expressed in any developmental process is feasible using this system.
The High chlorophyll fluorescence106 (Hcf106) gene in maize is required for chloroplast membrane biogenesis, and the hcf106-mum1 allele is caused by the insertion of a Robertson's Mutator Mu1 element into the promoter of the gene. Seedlings homozygous for hcf106-mum1 are pale green and die 3 weeks after germination, but only in the presence of Mutator activity conferred by active, autonomous Mu regulatory transposons elsewhere in the genome. When Mutator activity is lost, the mutant phenotype is suppressed, and homozygous plants have an almost wild-type phenotype. To isolate derivative alleles at the hcf106 locus that no longer require Mutator activity for phenotypic expression, we have developed a method for site-selected transposon mutagenesis in maize. This procedure, first described for Caenorhabditis elegans and Drosophila, involves using polymerase chain reaction (PCR) to screen pools of individuals for insertions and deletions in genes of known sequence. Pools of seedlings segregating for the progenitor allele hcf106-mum1 were screened by PCR for insertions and deletions associated with Robertson's Mutator. In a 360-bp target region, two new insertions and one deletion were identified in only 700 Mu-active gametes screened. One of the insertions was in the progenitor hcf106-mum1 allele and the other was in the wild-type allele, but all three new alleles were found to have break-points at the same nucleotide in the first intron. Unlike the hcf-106-mum1 progenitor allele, the deletion and one of the insertions conferred pale green seedling lethal phenotypes in the absence of mutator activity. However, the second insertion had a weak, viable phenotype under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
Transposable elements from the Robertson's Mutator family are highly active insertional mutagens in maize. However, mutations caused by the insertion of responder (non-autonomous) elements frequently depend on the presence of active regulator (autonomous) elements for their phenotypic effects. The hcf106::Mu1 mutation has been previously shown to depend on Mu activity in this way. The dominant Lesion-mimic 28 mutation also requires Mu activity for its phenotypic effects. We have used double mutants to show that the loss of Mu activity results in the coordinate suppression of both mutant phenotypes. This loss can occur somatically resulting in large clones of cells that have a wild-type phenotype. Autonomous and non-autonomous Mutator elements within these clones are insensitive to digestion with methylation-sensitive enzymes, suggesting extensive methylation of CG and non-CG cytosine residues. Our data are consistent with the sectors being caused by the cycling of MuDR regulatory elements between active and inactive phases. The pattern of sectors suggests that they are clonal and that they are derived from the apical cells of the vegetative shoot meristem. We propose that these cells are more likely to undergo epigenetic loss of Mu activity because of their longer cell division cycle during shoot growth. Coordinate suppression of unlinked mutations can be used to perform mosaic analysis in maize.
The Mu transposons of the Robertsons's Mutator transposable element system in maize are unusual in many respects, when compared to the other known plant transposon systems. The excision of these elements occurs late in somatic tissues and very rarely in the germ line. Unlike the other plant transposons, there is no experimental evidence directly linking Mu element excision and integration. We have analyzed the excision products generated by a Mu1 transposon inserted into the bronze 1 locus of maize. We find that the excision products or 'footprints' left by the Mu1 element resemble those of the other plant transposable elements, rather than those of the animal transposable element systems. We also find some novel types of footprints resembling recombinational events. We suggest that the Mu1 element can promote intrachromosomal crossovers and conversions near its site of insertion, and that this may be another mechanism by which transposons can accelerate the evolution of genomes.
Many transposable elements in maize alternate between active and inactive phases associated with the modification of their DNA. Elements in an inactive phase lose their ability to transpose, their ability to excise from reporter alleles and, in some cases, their ability to enhance or suppress mutant phenotypes caused by their insertion. The maize mutant hcf106 is a recessive pale green seedling lethal caused by the insertion of the transposable element Mu1. We show that the hcf106 mutant phenotype is suppressed in lines that have lost Mu activity. That is, homozygous hcf106 seedlings are dark green and viable when transposable elements belonging to the Robertson's Mutator family are modified in their terminal inverted repeats, a diagnostic feature of inactive lines. This property of the mutant phenotype has been used to follow clonal leaf sectors containing modified Mu elements that arise from single somatic cells during plant development. The distribution of these sectors indicates that epigenetic switches involving Mu DNA modification occur progressively as the meristem ages.
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The production of hydrolytic enzymes in the germinating cereal grain is considered as a model for plant cell differentiation. Recent literature is reviewed which suggests that gibberellins are involved in this process, but in a less straightforward manner than considered previously. It seems likely that only a subfraction of gibberellin is active and that production of this gibberellin is actually in the hydrolase-producing cells. These include aleurone cells and also the scutellar epithelial cells. At the intracellular level the action of gibberellin results in the accumulation of alpha-amylase mRNA and also mRNA for other unidentified proteins, referred to as gibb-ons. The alpha-amylase mRNAs are transcribed from two distinct gene families. The pattern of expression of alpha-amylase and gibb-on mRNAs is consistent with a common gibberellin-stimulated mechanism of control of for all of these genes. However it seems likely from experiments with ABA and from observations on gibb-on gene expression in non-aleurone cells that gibberellin does not have a role determining the specificity of gene expression, but rather acts as a general stimulator of mRNA accumulation.