Ac-immobilized, a stable source of Activator transposase that mediates sporophytic and gametophytic excision of Dissociation elements in maize

We have identified and characterized a novel Activator (Ac) element that is incapable of excision yet contributes to the canonical negative dosage effect of Ac. Cloning and sequence analysis of this immobilized Ac (Ac-im) revealed that it is identical to Ac with the exception of a 10-bp deletion of sequences at the left end of the element. In screens of approximately 6800 seeds, no germinal transpositions of Ac-im were detected. Importantly, Ac-im catalyzes germinal excisions of a Ds element resident at the r1 locus resulting in the recovery of independent transposed Ds insertions in approximately 4.5% of progeny kernels. Many of these transposition events occur during gametophytic development. Furthermore, we demonstrate that Ac-im transactivates multiple Ds insertions in somatic tissues including those in reporter alleles at bronze1, anthocyaninless1, and anthocyaninless2. We propose a model for the generation of Ac-im as an aberrant transposition event that failed to generate an 8-bp target site duplication and resulted in the deletion of Ac end sequences. We also discuss the utility of Ac-im in two-component Ac/Ds gene-tagging programs in maize.     

Expression of tandem gene fusions in transgenic tobacco plants.

We have studied the expression of four sets of tandem gene fusions in transgenic tobacco plants. This was to determine if the problem of between-transformant variability in expression of introduced genes could be overcome by using a linked reference gene as a co-ordinately expressed control. Tandem gene fusions containing identical 5' flanking regions (SSU301-ocs with either SSU301-cat or SSU301-SSU911) were not co-ordinately expressed in the transgenic tobacco plants whereas the tandem gene fusions containing similar but not identical 5' flanking regions (SSU301-ocs with SSU911-cat or SSU911-SSU301) were co-ordinately expressed. The lack of co-ordinate expression of some of the tandem gene fusions appears to be partially explained by absence of the corresponding genomic DNA segments in the transgenic plants.     

Somatic and germinal activities of maize Activator (Ac) transposase mutants in transgenic tobacco

The properties have been investigated of two deletion derivatives of the transposase protein (TPase) of maize transposable element Ac in transgenic tobacco. The wild-type and mutant TPases were expressed as fusions to the cauliflower mosaic virus 35S promoter. A deletion of 102 amino acids from the N-terminus, TPase(103–807), induces Ds excisions from a SPT::Ds reporter locus with a higher frequency than the wild-type TPase. The increased transpositional activity of TPase(103–807) is a dominant trait, as seedlings coexpressing truncated and wild-type TPase show the characteristic TPase(103–807) variegation pheno-type. A transpositionally inactive TPase deletion derivative lacking 188 amino acids from the N-terminus inhibits the transpositional activity of the wild-type TPase.     

Fusion of the inducible promoter of the PR-1a gene to the Activator transposase gene can transactive excision of a non-autonomous transposable element by external and by internal stimuli

The open reading frame coding for the transposase gene of the maize transposon Activator (Ac) was expressed in transgenic tobacco plants under the control of the promoter of the inducible gene for pathogenesis-related protein 1a (PR-1a). Excision of a non-autonomous transposable element (Ds) from chimeric β-glucuronidase (GUS) and luciferase reporter gene constructs was employed to analyze the induction of the Ac transposase by external and by internal stimuli. Applying the GUS histochemical assay, Ds excision events were detected in leaves, stems, and roots after treatment of regenerating shoots with salicylic acid (SA). Varying the SA induction procedure led to different Ds excision patterns in leaves and in roots. Furthermore, Ds excision events were also observed in non-treated, older transgenic plants in the green leaves, but not in germinal cells. Thus, the PR-1a promoter/Ac transposase gene fusion, together with the improved methods for induction of this chimeric gene, may provide a valuable tool for studying basic mechanisms of Ac transposition and for developing modified transposable element systems suitable for gene tagging in higher plants.     

Methylation pattern of Activator transposase binding sites in maize endosperm.

The maize transposable element Activator (Ac) transposes after replication from only one of the two daughter chromatids. It has been suggested that DNA methylation in conjunction with methylation-sensitive transposase binding to DNA may control the association of Ac transposition and replication. We present here a detailed genomic sequencing analysis of the cytosine methylation patterns of the transposase binding sites within both Ac ends in the wx-m9::Ac allele, where Ac is inserted into the tenth exon of the Waxy gene. The Ac elements in wx-m9::Ac kernels exhibit intriguing methylation patterns and fall into two distinct groups. Approximately 50% of the elements are fully unmethylated at cytosine residues through the 256 nucleotides at the 5' end (the promoter end). The other half is partially methylated between Ac residues 27 and 92. In contrast, at the 3' end, all Ac molecules are heavily methylated between residues 4372 and 4554. The more internally located Ac sequences and the flanking Waxy DNA are unmethylated. Although most methylated cytosines in Ac are in the symmetrical CpG and CpNpG arrangements, nonsymmetrical cytosine methylation is also common in the hypermethylated regions of Ac. These results suggest a model in which differential activation of transposon ends by hemimethylation controls the chromatid selectivity of transposition and the association with replication.     

Preferential transposition of the maize element Activator to linked chromosomal locations in tobacco.

The autonomous maize transposon Activator (Ac) has been used in maize for gene isolation by tagging and may prove similarly useful in other species. To test the feasibility of gene tagging with heterospecific transposons, we have examined three key genetic properties of a slightly modified Ac in tobacco. First, we show that frequencies of germinal excision of this Ac element from the antibiotic resistance gene streptomycin phosphotransferase can be comparable with or slightly lower than in maize. Second, we show that about half of the progeny carrying a germinal excision product also carry a transposed Ac. Last, we have mapped transposed Ac locations relative to the streptomycin transferase gene excision product and have shown that as in maize Ac in tobacco preferentially transposes to genetically linked sites.     

Promoter analysis of iron-deficiency-inducible barley IDS3 gene in Arabidopsis and tobacco plants.

Under conditions of iron deficiency, graminaceous plants induce the expression of genes involved in the biosynthesis of mugineic acid family phytosiderophores. We previously identified the novel cis-acting elements IDE1 and IDE2 (iron-deficiency-responsive element 1 and 2) through promoter analysis of the barley (Hordeum vulgare L.) iron-deficiency-inducible IDS2 gene in tobacco (Nicotiana tabacum L.). To gain further insight into plant gene regulation under iron deficiency, we analyzed the barley iron-deficiency-inducible IDS3 gene, which encodes mugineic acid synthase. IDS3 promoter fragments were fused to the beta-glucuronidase (GUS) gene, and this construct was introduced into Arabidopsis thaliana L. and tobacco plants. In both Arabidopsis and tobacco, GUS activity driven by the IDS3 promoter showed strongly iron-deficiency-inducible and root-specific expression. Expression occurred mainly in the epidermis of Arabidopsis roots, whereas expression was dominant in the pericycle, endodermis, and cortex of tobacco roots, resembling the expression pattern conferred by IDE1 and IDE2. Deletion analysis revealed that a sequence within -305 nucleotides from the translation start site was sufficient for specific expression in both Arabidopsis and tobacco roots. Gain-of-function analysis revealed functional regions at -305/-169 and -168/-93, whose coexistence was required for the induction activity in Arabidopsis roots. Multiple IDE-like sequences were distributed in the IDS3 promoter and were especially abundant within the functional region at -305/-169. A sequence moderately homologous to that of IDE1 was also present within the -168/-93 region. These IDE-like sequences would be the first candidates for the functional iron-deficiency-responsive elements in the IDS3 promoter.     

Variegation patterns caused by excision of the maize transposable element Dissociation (Ds) are autonomously regulated by allele-specific Activator (Ac) elements and are not due to trans-acting modifier genes

The Ac elements present in the unstable wxm7 and wx-m9 alleles of maize trigger different patterns of Ds excision in trans. To determine whether this differential regulation is a feature of the Ac alleles themselves or is mediated by genetically distinct factors, maize plants heterozygous for the wx-m7 and wx-m9 alleles were crossed to tester strains homozygous for Ds reporter alleles. Kernels showing the variegation pattern characteristic for the Ac elements carried in the wx-m7 and wx-m9 alleles were found to be present in the ratios expected from the genetic constitution of the strains. The aleurone variegation caused by excision of the Ds reporter element and the endosperm variegation caused by excision of Ac from the wx-m7 and wx-m9 alleles themselves segregated with the original wx-m alleles. In addition, stable Wx and wx derivatives of wx-m9 that have lost Ac no longer exert any trans effect on the wx-m7 allele (and vice versa). Therefore it is concluded that the observed variegation patterns are autonomously determined by specific trans effects of the particular Ac element.     

Evidence for type II phytochrome-induced rapid signalling leading to cab::luciferase gene expression in tobacco cotyledons

We studied the activation of cab gene expression by phytochrome-induced intercellular signalling and report insights into the mechanism of induction and outspread of a plant internal signal. By micro-beam irradiation techniques and use of a photon-imaging charge-coupled device (CCD) camera system we monitored cab::luciferase reporter gene expression in cotyledons of transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) plants. We found that (i) the photoreceptor triggering intercellular signalling and reporter-gene expression is type II but not type I phytochrome, (ii) phytochrome in its far-red-absorbing (Pfr) form is necessary for the induction but not for the outspread of the signalling, (iii) red/far-red reversibility is restricted to the red-irradiated cells, and (iv) the phytochrome-induced signal spreads rapidly throughout the cotyledon and reaches its target cells within minutes. Received: 26 June 2000 / Accepted: 25 August 2000     

Use of gene fusions to determine the orientation of gene phoA on the Escherichia coli chromosome.

We present genetic evidence which demonstrates that the phoA gene is transcribed in the clockwise direction on the Escherichia coli chromosome, in contrast to an earlier proposal. Our conclusion is based on analysis of various genetic fusions between the lac operon and the phoA gene.     

Selection of lac gene fusions in vivo: ompR-lacZ fusions that define a functional domain of the ompR gene product.

We describe a simple method for selecting Escherichia coli mutants that carry gene fusions between a cloned gene and lacZ. We test this technique with the ompR gene, which codes for a positive regulatory factor in porin synthesis. A number of OmpR-LacZ hybrid proteins are examined, and several unusual phenotypes associated with these protein fusions are described. Evidence is presented to support the two-domain model for ompR proposed previously (Hall and Silhavy, J. Mol. Biol. 151:1-15). In addition, one of the ompR-lacZ fusions exhibits a dominant OmpR- phenotype. The utility of isolating a series of lacZ gene fusions to any target gene is discussed.