Showing papers by "Tony A. Kavanagh published in 2002"

NAN fusions: a synthetic sialidase reporter gene as a sensitive and versatile partner for GUS.

Abstract: GUS continues to be the reporter of choice for many gene fusion applications, due to the unparalleled sensitivity of the encoded enzyme and the ease with which it can be quantified in cell-free extracts and visualized histochemically in cells and tissues. A compatible and functionally equivalent reporter gene would facilitate dual promoter studies and internal standardization of expression analyses in the same plant. A search for a candidate enzyme activity not found in plants, which might form the basis of a novel GUS-compatible reporter system, led us to investigate nanH, a Clostridium perfringens gene which encodes the so-called 'small' cytoplasmic sialidase. Expression of the native, AT-rich nanH gene in transgenic plants did not, however, result in detectable sialidase activity. For this reason, a codon-optimized derivative, NAN, was synthesized which possesses a GC content similar to that found in highly expressed plant genes. NAN enzyme activity was expressed at high levels in both stably and transiently transformed cells, possessed kinetic and stability properties similar to those of GUS, and showed optimal activity in GUS buffer. Moreover, NAN and GUS activity could be visualized simultaneously in polyacrylamide gels using the corresponding methylumbelliferone-based substrates, and in whole seedlings and tissue sections using the histochemical substrates 5-bromo-4-chloro-3-indolyl alpha-d-N-acetylneuraminic acid (X-NeuNAc) and 5-bromo-6-chloro-3-indolyl beta-d-glucuronide (X-GlucM), respectively.

Evolutionary Re-organisation of a Large Operon in Adzuki Bean Chloroplast DNA caused by Inverted Repeat Movement

Abstract: We have sequenced two sections of chloroplast DNA from adzuki bean (Vigna angularis), containing the junctions between the inverted repeat (IR) and large single copy (LSC) regions of the genome. The gene order at both junctions is different from that described for other members of the legume family, such as Lotus japonicus and soybean. These differences have been attributed to an apparent 78-kb inversion that spans nearly the entire LSC region and which is present in adzuki and its close relative, the common bean. This 78-kb rearrangement broke the large S10 operon of ribosomal proteins into two smaller operons, one at each end of the LSC, without affecting the gene content of the genome. It disrupted the physical and transcriptional relationship between the six-gene rpl23-rpl14 cluster and the four-gene rps8-rpoA cluster that is conserved in most land plants. Analysis of the endpoints of the rearrangement indicates that it probably occurred by means of a two-step process of expansion and contraction of the IR and not by a 78-kb inversion.

Plastid genes transcribed by the nucleus-encoded plastid RNA polymerase show increased transcript accumulation in transgenic plants expressing a chloroplast-localized phage T7 RNA polymerase.

Abstract: A gene fusion encoding a plastid-targeted bacteriophage T7 RNA polymerase (T7RNAP) under the transcriptional control of the light-regulated promoter and the plastid-targeting signals of a ribulose-bisphosphate carboxylase/oxygenase (Rubisco) small-subunit (SSU) gene was introduced into the nuclear genome of Nicotiana tabacum (tobacco). Immunoblot analysis, in vitro transcription assays and protease treatment of isolated chloroplasts revealed that T7RNAP activity was localized within chloroplasts. RNA gel blot analyses showed a substantial increase in transcript abundance for several plastid genes that are normally transcribed by the nucleus-encoded plastid RNA polymerase (NEP) including rpoC1, rpl33, rps18, rps12, and clpP. By contrast, no significant changes were observed in the levels of psbD, 16SrDNA, and ndhA transcripts. These results suggest a possible direct or indirect T7RNAP-mediated enhancement of transcription of a subset of plastid genes that contain NEP promoters. Despite these alterations in plastid transcript levels, the plants showed no visible abberant phenotype.