Mosaic Virus 35S RNA Promoter Research Articles

Function of the hexameric sequence in the cauliflower mosaic virus 35S RNA promoter region

The hexameric sequence ACGTCA functions in transcriptional regulation of wheat histone genes. The cauliflower mosaic virus (CaMV) 35S RNA promoter has the same hexameric sequence, and mutation analyses confirmed that the hexamer contributed greatly to transcription from the 35S promoter when a test gene with this promoter was introduced into sunflower cells. Electrophoretic mobility shift assays revealed the existence of a nuclear protein(s) in sunflower cells which is homologous to the HBP-1b that has been identified as binding to the 35S promoter in wheat. These results provide evidence of the involvement of the hexameric sequence and the HBP-1b-like DNA binding protein(s) in transcription from the 35S promoter.

Replication of tomato golden mosaic virus DNA B in transgenic plants expressing open reading frames (ORFs) of DNA A: requirement of ORF AL2 for production of single-stranded DNA.

Tomato golden mosaic geminivirus has a genome of two single-stranded (ss) DNA components, A and B. An almost identical 'common' region in DNA A and DNA B is thought to contain sequence elements controlling replication and transcription. Hence investigation of sequences important for DNA replication by in vitro mutagenesis is complicated by possible effects on the transcription of genes for replication proteins. To overcome this problem, transgenic plants expressing open reading frames (ORFs) of DNA A from an enhanced cauliflower mosaic virus 35S RNA promoter were constructed and tested for their ability to support the replication of DNA B and DNA B mutants. The results show that plants transgenic for ORF AL1 are able to support the replication of the double-stranded (ds) forms of DNA B, but that ORF AL2 is required in addition to produce ssDNA B. ORFs AL3, BL1 or BR1 were not required for replication of ds or ssDNA B. To the best of our knowledge this is the first time that essential replication proteins of a geminivirus have been expressed constitutively from a plant genome without giving rise to replicating DNA A molecules, thereby allowing DNA B to replicate alone. Such transgenic plants should enable not only the mutational analysis of sequence elements within the replication origin region, but also the construction of a new generation of vectors for gene amplification in plants, based on a minimal virus replicon.

Stability and expression of bacterial genes in replicating geminivirus vectors in plants

Bacterial beta-glucuronidase (gus) and neomycin phosphotransferase (neo) genes were introduced into coat protein replacement vectors based on DNA A of tomato golden mosaic virus (TGMV). Recombinant gus and neo vectors up to 1.1 kbp larger than DNA A were shown to replicate stably in transgenic plants containing partial dimers (master copies) of the vectors integrated into their chromosomal DNA in the absence of DNA B. Beta-glucuronidase and neomycin phosphotransferase activities in independently transformed plants were proportional to the copy number of the double-stranded forms of the vector. Deletion analysis has shown that an essential part of the TGMV coat protein promoter, including a TATA box, lies within 76 nt upstream of the initiation codon of the gene. An increase in expression of a neo gene was obtained by replacing this 76 nt sequence by an 800 nt sequence containing a cauliflower mosaic virus 35S RNA promoter with no effect on the ability of the vector to replicate or on its stability in transgenic plants. Systemic infection of plants by agroinoculation with TGMV vectors larger than DNA A in the presence of DNA B resulted in deletions in the vector DNA in some, but not all, plants. Possible reasons for vector instability in systemically infected plants, and vector stability in transgenic plants containing master copies of the vector, are discussed.

Functional regions of the cauliflower mosaic virus 35S RNA promoter determined by use of the firefly luciferase gene as a reporter of promoter activity

The cauliflower mosaic virus (CaMV) 35S RNA promoter has been dissected and examined in a transient expression system using the firefly luciferase gene as a reporter of promoter activity. Deletion analysis has shown that the 35S RNA promoter is composed of at least three regions-distal, medial, and proximal-which are essential for activity. The distal region contains three smaller elements homologous to the simian virus 40 "core" enhancer element, the medial region possesses a CCAAT-like box, and the proximal region contains a TATA box. A DNA segment encompassing the distal region is capable of activating the CaMV 35S core promoter in an orientation-independent, but not position-independent, fashion. The distal region can also activate a heterologous weak promoter, the CaMV 19S RNA promoter, albeit not to the high levels of the 35S RNA promoter. Multimers of the distal region are able to activate the 35S RNA promoter core to even greater levels of expression than the native 35S promoter. These experiments demonstrate that elements outside the boundaries of the core promoter (composed of proximal and medial elements) are recognized in a plant cell transient expression system.

Transient and Stable Expression of the Firefly Luciferase Gene in Plant Cells and Transgenic Plants

The luciferase gene from the firefly, Photinus pyralis, was used as a reporter of gene expression by light production in transfected plant cells and transgenic plants. A complementary DNA clone of the firefly luciferase gene under the control of a plant virus promoter (cauliflower mosaic virus 35S RNA promoter) was introduced into plant protoplast cells (Daucus carota) by electroporation and into plants (Nicotiana tabacum) by use of the Agrobacterium tumefaciens tumor-inducing plasmid. Extracts from electroporated cells (24 hours after the introduction of DNA) and from transgenic plants produce light when mixed with the substrates luciferin and adenosine triphosphate. Light produced by the action of luciferase was also detected in undisrupted leaves or cells in culture from transgenic plants incubated in luciferin and in whole transgenic plants "watered" with luciferin. Although light was detected in most organs in intact, transgenic plants (leaves, stems, and roots), the pattern of luminescence appeared to reflect both the organ-specific distribution of luciferase and the pathway for uptake of luciferin through the vasculature of the plant.

Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector.

Two bacterial antibiotic resistance genes, one coding for the neomycin phosphotransferase (NPT I) from Tn903, and the other coding for the chloramphenicol acetyltransferase from Tn9 were used as plant selectable markers. Both genes were introduced into the Nicotiana tabacum genome in a new plant expression vector, using the direct gene transfer method. The vector pDH51, used in these experiments contains a plant expression unit as a movable cassette, consisting of the strong cauliflower mosaic virus (CaMV) 35S RNA promoter and transcription terminator separated by a polylinker containing several unique restriction sites.