Epigenetic instability and trans-silencing interactions associated with an SPT::Ac T-DNA locus in tobacco.

Progeny of tobacco line 2853.6, which carries a streptomycin phosphotransferase (SPT) gene interrupted by the maize element Activator (Ac), were selected for streptomycin resistance (Spr) because of germinal Ac excision. Some events gave rise to Spr alleles that were unstable and exhibited a mottled phenotype on streptomycin-containing medium due to somatic loss of SPT function. This instability was most pronounced in one particular line, Spr12F. Other Spr alleles rarely exhibited silencing of SPT. Streptomycin-sensitive, homozygous Spr12F plants were recovered, and crosses were performed with other, more stable Spr lines. A high proportion of the resulting heterozygous progeny were silenced for SPT expression. The silenced state was heritable even after the Spr12F allele segregated away. No correlation could be made between silencing and methylation of the SPTgene. Structural analysis of allele Spr12F showed that the SPT gene from which Ac had excised was flanked by direct repeats of Ac. A search was carried out among 110 additional Spr alleles for new independent unstable alleles, and four were identified. All of these alleles also carried an SPT gene flanked by direct repeats of Ac. Thus, there is a strong correlation between this structure and instability of SPT expression.

The Frequency and Degree of Cosuppression by Sense Chalcone Synthase Transgenes Are Dependent on Transgene Promoter Strength and Are Reduced by Premature Nonsense Codons in the Transgene Coding Sequence.

By comparing the effects of strong and weak promoters that drive sense chalcone synthase (Chs) transgenes in large populations of independently transformed plants, we show here that a strong transgene promoter is required for high-frequency cosuppression of Chs genes and for production of the full range of cosuppression phenotypes. In addition, sense Chs transgenes driven by a cauliflower mosaic virus 35S promoter possessing a single copy of the upstream activator region (UAR) were found to produce a significantly lower degree of cosuppression than they did when the transgene promoter possessed two or four copies of the UAR. It has been shown elsewhere that 35S promoter strength increases with increasing UAR copy number. Frameshift mutations producing early nonsense codons in the Chs transgene were found to reduce the frequency and the degree of cosuppression. These results suggest that promoter strength and transcript stability determine the degree of cosuppression, supporting the hypothesis that sense cosuppression is a response to the accumulation of transcripts at high concentrations. This conclusion was shown to apply to single-copy transgenes but not necessarily to inversely repeated transgenes. The results presented here also have significance for efficient engineering of cosuppression phenotypes for use in research and agriculture.

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.

Aberrant Transpositions of Maize Double Ds-Like Elements Usually Involve Ds Ends on Sister Chromatids.

McClintock's analysis of chromosome-breaking Dissociation (Ds) elements in maize demonstrated that sister chromatids fuse at the position of Ds, forming a dicentric chromosome and an acentric fragment. In tobacco, Ds left and right ends in direct orientation (that is, half a double Ds) are sufficient to promote Activator-dependent marker gene loss. We present here a detailed analysis of germinally inherited rearrangements promoted by "half double Ds" elements and a characterization of rearrangements that involve inversion of the segment between the Ds ends and/or deletion of a segment adjacent to the Ds construct. The results support a model in which chromosome breakage promoted by these elements, and presumably by double Ds elements, involves Ds ends on sister chromatids.

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.

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.

Sequence of three bronze alleles of maize and correlation with the genetic fine structure.

The genomic sequences of three bronze alleles from Zea mays, Bz-McC, Bz-W22 and bz-R, are presented together with their flanking sequences. The bronze locus encodes UDPglucose flavonoid glucosyl-transferase (UFGT), an anthocyanin biosynthetic enzyme. The wild-type alleles Bz-McC and Bz-W22 condition purple phenotypes in the seed and plant, while bz-R conditions a bronze color. A full length cDNA corresponding to the Bz-McC allele was cloned and sequenced. Primer extension and RNase protection experiments were used to verify the 5' end of the bronze transcript. The Bz-McC allele has a 1416-bp coding region, a 100-bp intron and an approximately 83-bp 5' leader. Upstream of the message initiation site the sequences CTAACT and AATAAA occupy the positions where the eukaryotic consensus CCAAT and TATA boxes are normally found. The alleles Bz-McC and bz-R each have different large insertions with characteristics of transposable elements in their 5' flanking regions. The bz-R allele is distinguished by a 340-bp deletion starting within the intron and including 285 bp of the second exon. The Bz-McC and Bz-W22 isoalleles are known to differ in two genetically defined locations. The uts and uqv sites from the Bz-McC allele condition, respectively, lowered thermostability for the UFGT enzyme and increased amount of UFGT activity when compared with the corresponding sites in the Bz-W22 allele. The uts site maps to a region of the gene encoding two adjacent amino acid differences, either or both of which might alter the thermostability of the UFGT enzyme. The difference in UFGT levels conditioned by the uqv site is shown here to be correlated with variation in the bronze mRNA level. A likely cause of this decreased bronze mRNA level in Bz-W22 is a 6-bp duplication near the sequence CTAACT located 74 bp upstream of the bronze message initiation site. This region is therefore tentatively identified as the uqv site.

Stability of deletion, insertion and point mutations at the bronze locus in maize.

Phenotypic revertants from several kinds of mutations, including deletions, have been detected by pollen analysis at the wx and Adh loci in maize. Mutations in these genes give phenotypic revertants with median frequencies of 0.7 and 0.5×10(-5), respectively. However, the nature of such revertants can only be analyzed following their recovery from conventional matings. In the current study large seed populations derived from crosses involving several bz (bronze) locus mutations in maize were examined for reversion to a Bz (purple) expression. Deletion, insertion and point mutations were included in the study. Principally, over 2 million gametes of the bz-R mutation, which is shown here to be associated with a 340 base pair deletion within the transcribed region of the gene, have been screened for reversion. No revertants from it or any of the other bz mutations have been recovered, even though a total of almost 5 million gametes from homoallelic crosses have been examined to date. Results from seed analysis are discussed in reference to those from pollen analysis in maize.