Timothy C. Hall

Phaseolin gene from bean is expressed after transfer to sunflower via tumor-inducing plasmid vectors.

Sequences coding for the bean seed protein phaseolin were inserted into transferred DNA regions of tumor-inducing plasmids. Constructions were devised in which the coding region of phaseolin was fused in the correct reading frame with the coding region of octopine synthase and placed under the transcriptional control of the octopine synthase promoter. Other plasmids were prepared to permit expression of the phaseolin-encoding sequences from the flanking phaseolin promoter region. The RNA transcribed in sunflower cells transformed with these constructions was characterized by hybridization procedures, SI nuclease mapping, and by translation in vitro of extracted RNA. These tests showed that the genomic intervening sequences were correctly excised. Immunoreactive phaseolin polypeptides were detected by enzyme-linked immunosorbent assay and by antibody hybridization to electrophoretically separated protein extracts of sunflower tissues isolated from crown gall tumors and of transformed sunflower cells grown in tissue culture. These results demonstrate the expression of a plant gene after transfer to a taxonomically distinct botanical family.

Transgene silencing in monocots

Plant gene silencing was originally thought to be a quirk of transformation procedures, but is now recognized to be a facet of vitally important gene regulatory systems, present in all organisms. Monocot plants, especially the grasses, play a foremost role in the agricultural economy of all nations, and their biotechnological manipulation offers great potential for both developed and developing countries. Here, we review reported instances of transgene silencing in monocots and relate the processes of transcriptional and post-transcriptional gene silencing (TGS, PTGS) in perspective to the rapidly burgeoning knowledge of these phenomena in many organisms. Recent findings include the involvement of an RNA-dependent RNA polymerase and a nuclease in PTGS systems and the close relationship between methylation and chromatin structure in TGS events.

Histochemical analysis of CaMV 35S promoter-β-glucuronidase gene expression in transgenic rice plants

The cauliflower mosaic virus promoter is commonly used to drive transcription of chimeric genes in transgenic plants, including the cereals. To determine the tissue and cell types of cereal plants that the promoter functions in, transgenic rice plants containing a CaMV 35S promoter/GUS chimeric gene were analyzed for GUS activity. Insertion of a 35S/GUS chimeric gene at low copy number into chromosomal DNA of plants regenerated from electroporated protoplasts was confirmed by gel blot hybridization analysis of uncut and endonuclease-digested DNA. Quantitative measurement showed that GUS activity was some tenfold higher in rice leaves than in tobacco leaves [8] whereas activities obtained for rice roots were similar to those reported for tobacco roots. Histochemical localization of GUS activity confirmed that the CaMV 35S promoter functions in cells of the leaf epidermis, mesophyll and vascular bundle. It is also active in the cortex and vascular cylinder of the root, but only marginally active in the root epidermis. The generally similar distribution and levels of GUS activity obtained in differentiated tissue of stably transformed rice plants indicates the value of the CaMV 35S promoter as a positive control for studies in gene activity in transgenic monocots and dicots.

Agrobacterium-mediated transformation of Javanica rice

Difficulties frequently encountered using direct DNA transfer methods for transformation of Javanica varieties of rice (Oryza sativa L.) have limited the application of biotechnology to these varieties. We now reportAgrobacterium-mediated transformation of Javanica cultivars Gulfmont and Jefferson that are, respectively, widely used or about to enter commercial cultivation in the southern USA. Vigorous, phenotypically normal, fertile plants expressing both the selectable marker and the gene of interest were obtained. Southern analysis showed that only one or two copies of the T-DNA insert were present. Sequence analysis of right border fragments of one line confirmed that insertion was into a coding region of rice nuclear DNA. This analysis also revealed the presence of relatively short regions of permuted T-DNA border sequences, similar to those found afterAgrobacterium-mediated transformation of dicots. Progeny analysis of lines bearing two copies showed co-segregation, indicating that they were located relatively closely on the same chromosome. The introduced genes were transmitted to the R1 and R2 generations in a Mendelian fashion, confirming the suitability of this approach for biotechnological improvement of elite rice cultivars.

Complete sequence of the binary vector Bin 19

Despite the widespread use of Bin 19 as a vector for plant transformation, detailed sequence information on its T-DNA region has only recently become available. We now show that the non-T-DNA region, like the T-DNA region, contains several superfluous insertions and find that some functional elements may not contain optimal sequences. Knowledge of the complete 11 777 bp sequence will aid in the construction of exceptionally efficient derivative vectors of approximately half this size. Precise knowledge of restriction sites and removal of unnecessary sequences will facilitate plasmid manipulations and plant transformation.

Genetic variation in the subunits of globulin-1 storage protein of French bean

SummaryCharge 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,β48T, β48S, β47, γ45.5, γ45S, and γ45C, and two minor bands: γ46T and γ46S 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.

Genome intruder scanning and modulation systems and transgene silencing

The widespread occurrence of transgene inactivation in plants and classical cases of silencing of duplicated sequences in fungi suggest that all genomes contain defense systems that are capable of monitoring and manipulating intrusive DNA. Such DNA might be recognized by its structure, its sequence composition relative to that of its genomic environment and possibly by its disruption of normal biochemical functions. Although methylation, especially of repeated sequences, is widely associated with gene inactivation, other attributes, including chromatin modification, may be involved. Elimination of inactivated intrusive DNA (presently best documented for filamentous fungi) may also contribute to genomic defense mechanisms in plants. Stable integration and expression of introduced genes are essential for genetically engineered crops, and thus transformation constructs must be designed to avoid host surveillance processes.

Epigenetic transcriptional silencing and 5-azacytidine-mediated reactivation of a complex transgene in rice.

Despite a growing number of reports indicating non-Mendelian inheritance of transgene expression in monocots, no detailed description of the structure and stability of the transgene exists for transformants generated by direct DNA-transfer techniques, making the cause for these observations difficult to determine. In this paper we describe the complex organization of Btt cryIIIA and bar transgenes in rice (Oryza sativa L.) that displayed aberrant segregation in R1 progeny. Silencing rather than rearrangement of the bar gene was implicated because the herbicide-sensitive R1 plants had a DNA hybridization profile identical to that of the resistant R0 parent and R1 siblings. Genomic DNA analysis revealed substantial methylation of the Ubi1/bar sequences in silenced plants and, to a lesser degree, in herbicide-resistant plants, suggesting that the transgene locus was potentiated for silencing. Nuclease protection and nuclear run-on assays confirmed that silencing was due to transcriptional inactivation. Treatment of R2 progeny of silenced plants with 5-azacytidine resulted in demethylation of the Ubi1 promoter and reactivation of bar gene expression, demonstrating a functional relationship for methylation in gene silencing. These findings indicate that methylation-based silencing may be frequent in cereals transformed by direct DNA protocols that insert multiple, often rearranged sequences.

Viral protein synthesis in barley protoplasts inoculated with native and fractionated brome mosaic virus RNA.

When barley protoplasts were inoculated with brome mosaic virus (BMV) RNAs 1 and 2, there was a pronounced synthesis of the 110,000- and 100,000-dalton virally coded proteins. In contrast, there was no detectable synthesis of any viral proteins following inoculation with RNA 3 alone or RNA 4 alone. When RNAs 1 and 2 were recombined with RNA 3 in the inoculum, the profile of proteins synthesized was identical to that following inoculation with similar quantities of unfractionated BMV RNA; i.e., the 35,000-dalton virally coded protein and coat protein were synthesized in addition to the two high-molecular-weight viral polypeptides. RNAs 1 and 2 were shown not to be selectively bound (in preference to RNAs 3 or 4); hence, these data reveal that one or both of these RNAs encode proteins involved in early events of infection, perhaps replication.

Transfer RNA-like structures in viral genomes.

Publisher Summary The occurrence of aminoacylatable transfer RNA (tRNA)-like structures in several groups of plant viruses strongly suggests that they have a biological function. Indeed, in the case of brome mosaic virus (BMV), a relatively simple 3′-specific modification of the RNA results in loss of infectivity. However, currently, no metabolic role for the tRNA-like structures has been demonstrated, and the likelihood of functions related to translation or viral assembly processes seem to be remote. Thus, a role in transcription events currently appears to be the most attractive suggestion. This is despite the fact that other RNA viruses, such as pea enation mosaic virus, which lacks a functional tRNA-like 3′ structure or even a 3′ poly(A) sequence, are efficiently transcribed in their hosts. Because of the analogous levels of specificity for aminoacylation, despite their very different structures, it is possible that comparative studies of viral and tRNAs may yield an insight to the features of RNA, which permit aminoacylation to occur. Comparisons of infective properties of native and chemically modified forms of aminoacylatable viral RNAs may reveal if the tRNA structure is related to transcription or translation events; alternatively, they may reveal a novel metabolic process.