Modification of the histone proteins that form the core around which chromosomal DNA is looped has profound epigenetic effects on the accessibility of the associated DNA for transcription, replication and repair. The SET domain is now recognized as generally having methyltransferase activity targeted to specific lysine residues of histone H3 or H4. There is considerable sequence conservation within the SET domain and within its flanking regions. Previous reviews have shown that SET proteins from Arabidopsis and maize fall into five classes according to their sequence and domain architectures. These classes generally reflect specificity for a particular substrate. SET proteins from rice were found to fall into similar groupings, strengthening the merit of the approach taken. Two additional classes, VI and VII, were established that include proteins with truncated/ interrupted SET domains. Diverse mechanisms are involved in shaping the function and regulation of SET proteins. These include protein-protein interactions through both intra-and inter-molecular associations that are important in plant developmental processes, such as flowering time control and embryogenesis. Alternative splicing that can result in the generation of two to several different transcript isoforms is now known to be widespread. An exciting and tantalizing question is whether, or how, this alternative splicing affects gene function. For example, it is conceivable that one isoform may debilitate methyltransferase function whereas the other may enhance it, providing an opportunity for differential regulation. The review concludes with the speculation that modulation of SET protein function is mediated by antisense or sense-antisense RNA.

Miniature inverted repeat transposable elements (MITEs) are thought to be a driving force for genome evolution. Although numerous MITEs are found associated with genes, little is known about their function in gene regulation. Whereas the rice ubiquitin2 (rubq2) promoter in rice (Oryza sativa) line IR24 contains two nested MITEs (Kiddo and MDM1), that in line T309 has lost Kiddo, providing an opportunity to understand the role of MITEs in promoter function. No difference in endogenous rubq2 transcript levels between T309 and IR24 was evident using RT-PCR. However, promoter analysis using both transient and stably transformed calli revealed that Kiddo contributed some 20% of the total expression. Bisulfite genomic sequencing of the rubq2 promoters revealed specific DNA methylation at both symmetric and asymmetric cytosine residues on the MITE sequences, possibly induced by low levels of homologous transcripts. When methylation of the MITEs was blocked by 5-azacytidine treatment, a threefold increase in the endogenous rubq2 transcript level was detected in IR24 compared with that in T309. Together with the observed MITE methylation pattern, the detection of low levels of transcripts, but not small RNAs, corresponding to Kiddo and MDM1 suggested that RNA-dependent DNA methylation is induced by MITE transcripts. We conclude that, although Kiddo enhances transcription from the rubq2 promoter, this effect is mitigated by sequence-specific epigenetic modification.

Vectors were constructed for the isolation of random transcriptional and translational fi-glucuronidase gene fusions in plants. This system is based on the random integration of the transferred DNA (T-DNA) into the plant nuclear genome. The Escherichia coli .3-glucuronidase coding sequence without promoter, and also devoid of its ATG initiation site in the translational gene fusion vector, was inserted in the T-DNA with its 5' end at a distance of 4 base pairs from the right T-DNA border sequence. Transgenic plants can be selected by using a chimeric (P355-nptlI-3' ocs) kanamycinresistance gene present in the same T-DNA. Subsequent screening of these for 13-glucuronidase expression allows the identification of clones harboring a fusion of the fi-glucuronidase coding sequence with plant 5' regulatory sequences. In particular, the histochemical staining using 5-bromo-4-chloro-3-indolyl 3-Dglucuronic acid (X-GlcA) facilitates the detailed analysis of tissue or cell-type specificity of the gene fusions. T-DNA-mediated gene fusions were reported to occur at high frequency, independently of genome size (3). Therefore, this method can in principle be applied to all plant species that can be transformed efficiently by Agrobacterium. The small cruciferous plant Arabidopsis thaliana is an ideal model system for molecular genetics because of its small genome size, low repetitive DNA content, short generation time, and well-known genetics (9). Efficient Agrobacterium-mediated transformation of Arabidopsis has been described (10-13).The aim of the present work was to combine the advantages of T-DNA insertion mutagenesis and gus gene fusion technology. By transformation of Arabidopsis with the appropriate T-DNA vectors, gus fusions were obtained at a high frequency. Different types of organ-and tissue-specific GUS expression patterns were found. The usefulness ofthe system was further proved by the cloning of a DNA fragment mediating phloem-specific gene expression in homologous and heterologous systems. § MATERIALS AND METHODSBacterial Strains and Vector Constructions. DNA manipulations were performed as described (14). E. coli JM101 (15) and K514 (16) were used for plasmid transformation. E. coli NM430 (17) and MC1061 (18) were used for growing bacteriophage EMBL4. Plasmid pUC8 (19) was used for subcloning.The pGV1030 was derived from pGV943 (20) by deleting the unique Pst I/Sca I fragment of pGV943, thereby inactivating the 8-lactamase gene.The binary vectors were mobilized (21) Abbreviations: T-DNA, transferred DNA; X-GlcA, 5-bromo-4-chloro-3-indolyl 1-D-glucuronic acid.

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