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.

The genomes of many (+)-stranded RNA viruses, including plant viruses and alphaviruses, consist of polycistronic RNAs whose internal genes are expressed via subgenomic messenger RNAs. The mechanism(s) by which these subgenomic mRNAs arise are poorly understood. Based on indirect evidence, three models have been proposed: (1) internal initiation by the replicase on the (-)-strand of genomic RNA, (2) premature termination during (-)-strand synthesis, followed by independent replication of the subgenomic RNA and (3) processing by nuclease cleavage of genome-length RNA. Using an RNA-dependent RNA polymerase (replicase) preparation from barley leaves infected with brome mosaic virus (BMV) to synthesize the viral subgenomic RNA in vitro, we now provide evidence that subgenomic RNA arises by internal initiation on the (-)-strand of genomic RNA. We believe that this also represents the first in vitro demonstration of a replicase from a eukaryotic (+)-stranded RNA virus capable of initiating synthesis of (+)-sense RNA.

Recombinant phage X177.4 contains a gene for j3 phaseolin, a major storage glycoprotein of French bean seed. A 3.8-kilobase Bgl II-Bam}Il fragment containing the entire 1700-base-pair coding region, together with 863 base pairs of 5' and 1226 base pairs of 3' flanking sequence, was inserted into the A66 Ti plasmid of Agrobacterium tumefaciens and used to transform tobacco. The level of phaseolin in the seeds of plants regenerated from cloned tissue was 1000-fold higher than in other tissues. The molecular weight of the phaseolin RNA transcript in tobacco seeds was identical to that found in bean seeds. The phaseolin protein in tobacco seed was glycosylated and appeared to undergo removal of the signal peptide. However, a large proportion of the phaseolin was cleaved into discrete peptides. These same peptides were formed as phaseolin was degraded during tobacco seed germination. The phaseolin gene appeared to be inserted as a single copy, and the proportion of phaseolin per genome copy in tobacco seeds (up to 3% of the total embryo proteins) resembled that in the bean seeds (40% of total seed protein, expressed from about 14 copies per diploid genome). Furthermore, the transplanted gene was turned on during tobacco seed development, and its protein product, phaseolin, was localized in the embryonic tissues. Finally, the phaseolin gene was inherited as a Mendelian dominant trait in tobacco.The transfer of foreign genetic information to broad-leafed plants by means (4) and maintained on Murashige and Skoog medium (MS) without hormone supplement. The tissue was cloned by the feeder plate method (9) and then placed in liquid culture to induce shoot-stem elongation (10). The shoots were grafted (11) onto 6-to 8-week-old N. tabacum var. Xanthi plants and grown at 22°C with a 16-hr photoperiod. Flowers were self-pollinated, and the seeds were allowed to mature.Quantitative and Qualitative Protein Assays. Proteins were extracted and quantified as described (8). Phaseolin was quantitated in tissues by dot-immunobinding assay (12). Protein patterns were analyzed after fractionation on a 13% polyacrylamide gel (13) or by two-dimensional gel electrophoresis (14) followed by electrophoretic immunoblot analysis with polyclonal antiserum to phaseolin (15). Antigenantibody complexes were visualized by treating the filters with 251I-labeled Staphylococcus protein A, followed by autoradiography.For immunodetection of concanavalin A-bound proteins, tissue extracts were incubated with concanavalin ASepharose beads followed by elution of the bound fraction with 1% NaDodSO4. The bound and unbound fractions were subjected to NaDodSO4/polyacrylamide gel electrophoresis followed by immunoblot analysis as described earlier.Isolation of RNA and Blot-Hybridization Analysis. Total RNA was isolated from leaves as described (16). RNA from developing seeds was prepared by isolation of polysomes followed by phenol extraction of the polysome pellet (17 3320The publication costs of this article were defrayed in part by page charge pay...

The complete nucleotide sequences of the gene and the mRNA coding for a specific phaseolin type French bean major storage protein have been determined. Comparison of these sequences reveals a phaseolin gene structure consisting of 80 base pairs (bp) of 5' untranslated DNA, 1,263 bp of protein-encoding DNA which is interrupted by five intervening sequences (IVSI,72 bp; IVS2, 88 bp; IVS3, 124 bp; IVS4, 128 bp; and IVS5, 103 bp), and 135 bp of 3' untranslated DNA. Sequences characteristic of eukaryotic promoters "CCAAT" and "TATA" are present in the 5' flanking DNA, and the eukaryotic poly(A) addition signal A-A-T-A-A-A occurs 16 bp before the first nucleotide of poly(A). The derived amino acid sequence yields an amino acid composition and a molecular weight compatible with those found for the a-type phaseolin protein. Two regions that probably serve as carbohydrate-peptide linkage recognition sites have been identified. A region of highly hydrophobic amino acids at the NH2 terminus of the protein suggests the presence of a signal peptide in the newly synthesized phaseolin protein."Phaseolin" is the name of a group of polypeptides which make up the major storage glycoprotein in the seeds of French bean (Phaseolus vulgaris L.), representing about 50% of the total protein in mature seeds (1). One-dimensional NaDodSO4/ polyacrylamide gel electrophoresis of phaseolin isolated from cotyledons of the cultivar Tendergreen resolves three polypeptide bands-a, ,B, and y, of [51][52][53][47][48], and 43-46 kilodaltons (kDa), respectively (2, 3). All three polypeptides are encoded in 16S mRNA species, and these proteins accumulate rapidly in the developing seed cotyledon, beginning when the cotyledons are about 7 mm in length and continuing until the cotyledons reach 17-19 mm in length (4). Two-dimensional gel electrophoretic separation of phaseolin resolves five polypeptides, indicating charge and molecular weight heterogeneity in the phaseolin protein pool (2). Peptide mapping of these phaseolin proteins after proteolytic and chemical cleavages shows that all of these proteins are highly homologous (5, 6), suggesting that they may be encoded in a multigene family. Genomic blot analysis using a cloned phaseolin gene as probe confirms that phaseolin is indeed encoded in a. multigene family (unpublished data).We have previously reported (7) the isolation and partial nucleotide sequence of the phaseolin genomic clone AG-APVPh 177.4 (A177.4) and of a cloned phaseolin cDNA, AG-cpPVPhl (cDNA1) which contains about 40% of a phaseolin mRNA transcript (7).In this paper we report the complete nucleotide sequence determination of the phaseolin genomic clone A177. A177.4 and its subclone AGpPVPh7.2 (p7.2) have been described (7). Additional pBR322 subclones from A177.4 (used to facilitate DNA sequence determinations) are the 3.0-kilobase pair (kbp) EcoRI-BamHI subelone AG-pPVPh3.0 and the 3.8-kbp Bgl II-BamHI subclone AG-pPVPh3.8 (see Fig. 1, clone 177.4 for location of these subcloned regions). These subclones were construc...

Mutations affecting spatial and temporal regulation of a beta‐phaseolin gene encoding the major storage protein of bean (Phaseolus vulgaris) were analyzed by stable and transient transformation approaches. The results substantiate the value of transient assays for rapid determination of the functionality of cis‐acting sequences and the importance of stable transformation to identify tissue‐specific determinants. Spatial information is specified primarily by two upstream activating sequences (UAS). UAS1 (−295 to −109) was sufficient for seed‐specific expression from both homologous and heterologous (CaMV 35S) promoters. In situ localization of GUS expression in tobacco embryos demonstrated that UAS1 activity was restricted to the cotyledons and shoot meristem. A second positive domain, UAS2 (−468 to −391), extended gene activity to the hypocotyl. Temporal control of GUS expression was found to involve two negative regulatory sequences, NRS1 (−391 to −295) and NRS2 (−518 to −418), as well as the positive domain UAS1. The deletion of either negative element caused premature onset of GUS expression. These findings indicate combinatorial interactions between multiple sequence motifs specifying spatial information, and provide the first example of the involvement of negative elements in the temporal control of gene expression in higher plants.

Charge and molecular weight heterogeneity of globulin-1 (G1) polypeptides of the bean, Phaseolus vulgaris L., were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Different bean cultivars were classified into three groups: 'Tendergreen', 'Sanilac', and 'Contender' on the basis of their protein subunit composition. Nine distinct major bands: α51,α49, α48.5,β48(T), β48(S), β47, γ45.5, γ45(S), and γ45(C), and two minor bands: γ46(T) and γ46(S) were found to account for the three profiles seen on one-dimensional SDS-PAGE. Two-dimensional analysis revealed these eleven protein bands to be composed of a minimum of fourteen distinct protein subunits. The 'Tendergreen' and 'Sanilac' types differ in their G1 polypeptide composition. The protein patterns of the 'Contender' types are intermediate, containing many protein subunits found in the patterns of the 'Tendergreen' and 'Sanilac' types suggesting a genetic and evolutionary relationship.

SummaryThe phas promoter displays stringent spatial regulation, being very highly expressed during embryogenesis and completely silent during all phases of vegetative development in bean, Phaseolus vulgaris. This pattern is maintained in transgenic tobacco and, as shown here, Arabidopsis. Dimethyl sulphate in vivo footprinting analyses revealed that over 20 cis-elements within the proximal 295 bp of the phas promoter are protected by factor binding in seed tissues whereas none are bound in leaves. The hypothesis that this complex profile represents a summation of several module (cotyledon, hypocotyl, and radicle)-specific factor-DNA interactions has been explored by the incorporation of site-directed substitution mutations into 10 locations within the À295phas promoter. Only 2.6% of À295phas promoter activity remained after mutation of the G-box; the CCAAAT box, the E-box and the RY elements were also found to mediate high levels of expression in embryos. Whereas the CACA element has dual positive and negative regulatory roles, the vicilin box was identified as a strong negative regulatory element. The proximal (À70 to À64) RY motif was found to bestow expression in the hypocotyl while all the RY elements contribute to expression in cotyledons but not to vascular tissue expression during embryogenesis. RY elements at positions À277 to À271, À260 to À254, and À237 to À231 were found to orchestrate radicle-specific repression. The G-box appears to be the functional abscisic acid responsive element and the E-site may be a coupling element. The results substantiate the concept that autarkical cis-element functions generate modular patterning during embryogenesis. They also reflect the existence of both redundancy and hierarchy in cis-element interactions. Importantly, the virtually identical expression patterns observed for the two distantly related plants studied argue strongly for the generality of function for the observed factor-element interactions.

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