James J. English

Suppression of Virus Accumulation in Transgenic Plants Exhibiting Silencing of Nuclear Genes.

Homology-dependent gene silencing contributes to variation between transgenic plants with respect to transgene and/or endogenous gene expression levels. Recent studies have linked post-transcriptional gene silencing and virus resistance in plants expressing virus-derived transgenes. Using a potato virus X vector, we present three examples in which silencing of nonviral transgenes prevented virus accumulation. This effect was dependent on sequence homology between the virus and the silenced transgene. Analysis of potato virus X derivatives carrying bacterial [beta]-glucuronidase (GUS) sequences showed that the 3[prime] region of the GUS coding sequence was a target of the silencing mechanism in two independent tobacco lines. Methylation of the silenced GUS transgenes in these lines was also concentrated in the 3[prime] region of the GUS coding sequence. Based on this concurrence, we propose a link between the DNA-based transgene methylation and the RNA-based gene silencing process.

Effective vectors for transformation, expression of heterologous genes, and assaying transposon excision in transgenic plants.

Progress in plant molecular biology has depended heavily on the availability of effective vectors for plant cell transformation and heterologous expression. In this paper we describe the structures of a wide array of plasmids which have proved extremely effective in (a) plant transformation, (b) expression of heterologous genes and (c) assaying the activity of transposons in transgenic plants. Constructs that confer resistance to kanamycin, hygromycin, streptomycin, spectinomycin and phosphinotricin, or that confer β-glucuronidase (GUS) gene expression are presented. Binary vector constructs that carry polylinkers of the pUC and Bluescript types are also described. Plasmids that permit the expression of any heterologous reading frame from either nopaline synthase (nos) or octopine synthase (ocs) promoters, as well as the cauliflower mosaic virus 35S promoter, using either the nopaline synthase or octopine synthase 3′ polyadenylation sequences, are presented. These constructs permit a choice of orientation of the resulting transgene of interest, relative to the orientation of the selection marker gene. Most of the plasmids described here are publicly available.

Ectopic pairing of homologous DNA and post-transcriptional gene silencing in transgenic plants

Abstract Recent studies indicate that co-suppression and other types of post-transcriptional gene silencing in plants are initiated by homology-dependent pairing of transgene sequences. Once initiated, the silencing mechanism acts in the cytoplasm to prevent accumulation of all RNAs with homology to the transgene.

Analysis of the chromosomal distribution of transposon-carrying T-DNAs in tomato using the inverse polymerase chain reaction

We are developing a system for isolating tomato genes by transposon mutagenesis. In maize and tobacco, the transposon Activator (Ac) transposes preferentially to genetically linked sites. To identify transposons linked to various target genes, we have determined the RFLP map locations of Ac- and Dissociation (Ds)-carrying T-DNAs in a number of transformants. T-DNA flanking sequences were isolated using the inverse polymerase chain reaction (IPCR) and located on the RFLP map of tomato. The authenticity of IPCR reaction products was tested by several criteria including nested primer amplification, DNA sequence analysis and PCR amplification of the corresponding insertion target sequences. We report the RFLP map locations of 37 transposon-carrying T-DNAs. We also report the map locations of nine transposed Ds elements. T-DNAs were identified on all chromosomes except chromosome 6. Our data revealed no apparent chromosomal preference for T-DNA integration events. Lines carrying transposons at known map locations have been established which should prove a useful resource for isolating tomato genes by transposon mutagenesis.

A molecular genetic analysis of insertions in the bronze locus in maize

SummaryWe have undertaken a combined molecular and genetic analysis of the bronze (bz) locus in maize. The bz locus was isolated by a two-step approach involving sequences from the transposable elements Dissociation (Ds) and Activator (Ac). A restriction map of the bz region is presented, identifying the transcribed region and the direction of transcription. We have mapped the sites of two Ds insertions, bz-m2 (DI) and bz-m1, physically and genetically. bz-m2 (DI) is a 3.5 kb insertion in the transcribed region that appears to have arisen by an internal deletion in the 4.5 kb Ac insertion in the mutant bz-m2. bz-m1 is a 1.1 kb insert in the 5′ end of the gene. By correlating the genetic and physical maps, we have been able to orient the restriction map relative to the centromere in chromosome 9 and have obtained a rough estimate of the physical length of a unit of genetic recombination in a microregion of the maize genome.

High level expression of the Activator transposase gene inhibits the excision of Dissociation in tobacco cotyledons

A fusion of the strong cauliflower mosaic virus 35S promoter to the Activator (Ac) transposase (TPASE) gene does not trigger excision of Dissociation (Ds) continuously during tobacco cotyledon development, although once activated, the 35S promoter remains active throughout embryogeny. Epistasis studies where 35S:TPASE is in trans with later-acting fusions indicate that transient effectiveness for excision results from this fusion inhibiting its own action and that of other TPASE sources. Inhibition depends on the strength of TPASE expression, since fusions of the 35S promoter to a TPASE cDNA accumulate 30-fold lower amounts of TPASE mRNA than the 35S:TPASE gene fusion and do not inhibit excision. We discuss the role of TPASE levels in the curious relationship between Ac activity and Ac dosage in maize.

Deletions and Breaks Involving the Borders of the Ac Element in the bz-m2(Ac) Allele of Maize

The corn Ac-Ds transposable element (te) system, the first one described by McClintock (15,16), consists of the autonomous element Ac (Activator) and the nonautonomous element Ds (Dissociation). That is, even though both elements can transpose, only Ac can induce its own transposition and that of Ds. There is extensive evidence in the literature that the te Ac can undergo and mediate a series of mutational changes. One type of change described by McClintock (17–20) is mutation of Ac to Ds. The genetic and molecular analysis of mutant Ds derivatives from Ac has helped to define regions of Ac that specify transposition functions in maize (8,12,22). With the recent demonstration that Ac transposes in tobacco (1), we can expect a finer mutational resolution of the transposase function of Ac. In this paper, we describe other types of mutations, the analysis of which has allowed us to obtain information on different transposition properties of Ac.

Strategies for the Cloning of Genes in Tomato for Resistance to Fulvia Fulva

We are developing two alternative strategies to clone one or more of the Cf2, Cf4, Cf5, Cf9 or Cf11 genes of tomato (Lycopersicon esculentum) that confer resistance to Fulvia fulva (syn. Cladosporium fulvum). The first approach is tagging with the maize transposon Ac. Since Ac preferentially transposes to closely linked sites in both maize and tobacco, close linkage should improve tagging efficiency. We are therefore RFLP mapping T-DNAs carrying Ac which have been transformed into tomato using as probes inverse polymerase chain reaction (IPCR) amplified tomato sequences adjacent to the T-DNA ends. Similarly, we are mapping the Cf genes on the RFLP map, so that we can identify for further analysis those transformants with T-DNAs closely linked to Cf genes. The second strategy involves the development of a technique of subtractive cDNA cloning, in which cDNA from a Cm plant (plant with no detectable resistance genes) is used to subtract cDNA which is common to a near-isogenic Cf2 plant, leaving Cf2 specific cDNA which can be cloned, and used as a probe for differential screening.